DE69736351T4 - METHODS AND MEANS FOR INHIBITING CDK4 ACTIVITY - Google Patents

Abstract

p21<SUP>WAF1 </SUP>interacts with cyclin D1 and Cdk4. Peptide fragments of p21 inhibit the interaction and/or affect Cdk4 activity. The peptides, derivative peptides and non-peptidyl mimetics thereof are useful in affecting activity of Cdk4, such as RB phosphorylation and cellular proliferation, indicative of therapeutic usefullness in treatment of tumors and other hyperproliferative disorders. Assay and screening methods allow identification of such modulators, especially inhibitors, of Cdk4 activity.

Description

The present invention relates to substances and their therapeutic use, and more particularly to the identification of regions of p21 ^WAF1 that ^bind to cyclin-dependent kinases, particularly Cdk4 and / or cyclin D1, as well as substances, fragments and mimetics based on this region. The present invention also relates to pharmaceutical compositions comprising these molecules, as well as their use in therapeutic applications for the treatment of hyperproliferative disorders such. Cancer and psoriasis, and compositions comprising these molecules, and their use in applications related to growth in eukaryotic cells. The invention also relates to assay methods and means for identifying substances useful for interfering with the p21 / Cdk4 / cyclin interaction, and preferably inhibiting Cdk4 activity.

The tumor suppression function of p53 has been implicated in a DNA damage inducible cell cycle checkpoint pathway (Kastan et al., 1991) in which p53 in the damaged cells either arrested growth (Agarwal et al., (1995) )) or apoptosis (Clarke et al., 1993; Lowe et al., 1993; Merrit et al., 1994). The biochemical activity of p53, which is most closely associated with tumor suppression and growth arrest, involves ionizing, radiation-dependent activation of sequence-specific transcriptional activity (Kastan et al., 1991; Lu & Lane, 1993; Pietenpol et al. 1994)). p53 induces transcription in a number of genes whose products play a direct role in mediating growth arrest. These p53-inducible negative regulators of cell proliferation include: the cyclin-dependent kinase inhibitor (CKI), p21 ^WAF1 (El-Deiry et al., 1993; Harper et al., 1993; Xiong et al., 1993; Gu et al., (1993)); an apoptosis-promoting protein, Bax (Miyashita & Reed (1995)); the insulin growth factor binding protein IGF-BP3 (Buckbinder et al., 1995) and Gadd45 (Kastan et al., 1992), a powerful inhibitor of cell proliferation with a currently unclear biochemical function (Kearsey et al., 1995) ,

A frequent event in the development of human neoplasia is the inactivation of a DNA damage-inducible cell cycle checkpoint pathway regulated by p53 (Hollstein et al (1991); Lane (1992); Agrawal et al. (1995)). , A number of mechanisms may lead to functional inactivation of the p53 pathway, including: missense mutations within or deletions of the p53 gene, inactivation of the function of the wild-type p53 protein through interaction with the oncogenic cellular protein mdm-2 (Momand et al. (1992)) or the inability to use downstream effector molecules, such as , P21 ^WAF1 (Deng et al., 1995; Waldman et al., 1995).

The inventors' growing knowledge of the molecular mechanisms underlying mammalian cell transformation provides the opportunity to create appropriately created inhibitors of specific biochemical processes essential for uncontrolled cell proliferation or cancer. Recent developments have shown that the reactivation of the p53 pathway in some human tumors can theoretically be accomplished as follows: (i) activation of the biochemical function of mutant p53 protein (Halazonetis & Kandil (1993); Hupp et al. (1993)) possibly using small peptides, as is usually the case in drug design (Hupp et al. (1995)); (ii) disrupting the interaction of the oncogene mdm-2 and the wild-type p53 through the use of peptide-mimetic inhibitors of complex formations (Picksley et al., 1994); (iii) restoring or mimicking the function of the downstream effector molecule p21 ^WAF1 , which alone is capable of mediating growth suppression (El-Deiry et al., 1993; Eastham et al., 1995).

p21 ^WAF1 is an inhibitor of both the G1-cyclin dependent protein kinases (CDKs that control the progression from G1 to S phase) (Harper et al., 1995) and the nuclear antigen-proliferating cell protein (PCNA; DNA replication factor) (Florez-Rozas et al., 1994; Waga et al., 1994). Thus, inhibition of the function of either CDKs or PCNA theoretically provides two distinct pathways for the development of drug discovery ^programs based on the activity of p21 ^WAF1 . The PCNA-binding function of p21 ^WAF1 can be ^mimicked by a 20 amino acid ^peptide derived from the C-terminal domain of p21 ^WAF1 , and this peptide is sufficient to partially inhibit SV40 replication in vitro (Warbrick et al (1995)).

Despite its PCNA-binding role, the main function of the p21 ^WAF1 protein as a growth ^suppressor appears to be inhibition of the G1-cyclin-CDK complex (Chen et al., 1995; Harper et al., 1995; Luo et al. 1995); Nakanishi et al. (1995b)). Luo et al. (1995) reported that the N-terminal domain of p21, consisting of residues 1-75, would act as a CDK inhibitor in vitro and inhibit cyclin E-Cdk2.

The present invention relates to (i) the elucidation of the molecular mechanism of cyclin D1-Cdk4 complex ^inhibition by p21 ^WAF1 and (ii) the identification of peptidomimetics with p21 ^{WAF1-inhibitory} activity by studying the binding and inhibition properties of a range of synthetic peptides based on the amino acid sequence of p21 ^WAF1 . The inventors' studies have revealed that two peptides derived from the N-terminal domain of p21 ^{WAF1 have} biochemical activity; one peptide 4 (residues 46-65) forms a stable complex with Cdk4, but has no inhibitory activity, while a peptide 2 (residues 16-35) binds to cyclin D1 and Cdk4 activity binds with a I0.5 of 2 μM inhibits.

These data define a cyclin binding site on p21 ^WAF1 and suggest that a mechanism involved in the CDK inhibitory effect of p21 ^{WAF1 utilizes} binding to the cyclin subunit of the CDK holoenzyme. This led to the proposal of the inventors that p21 ^WAF1 could alter Cdk4 activity allosterically by conformational or (ii) disruption of cyclin-Cdk interaction or (iii) disruption of cyclin-Cdk-substrate interaction changes in the structure of cyclin Inhibit D1. Furthermore, the peptides based on the C-terminal sequence of p21 ^{WAF1 interact} with both cyclin D1 and Cdk4 and are potent inhibitors of Cdk4 activity, with one peptide (herein peptide 10) composed of residues 141-160 and one I has _0.5 of 0.1 μM. The inventors show that both of the inhibitory peptides bind to cyclin D1 and / or Cdk4 at the physiologically relevant sites and that they have a specificity that mimics that of full length p21 ^WAF1 . It is important to note that the effectiveness of the C-terminal peptide is improved by a single amino acid substitution (DA at position 149). The inventors have mapped the inhibitory component of this peptide using alanine mutation analysis and show that it differs from the PCNA interaction domain, which is also located in the C-terminal region of the p21 ^WAF1 protein.

Remarkably, a stretch of just five amino acids contains the Cdk4-inhibiting motif, and a single conservative mutation on either of the two hydrophobic amino acid residues completely abolishes the inhibitory effect of the peptide. These data have exciting implications for the mechanism of action of the p21 ^WAF1 protein and represent a starting point for a drug design program aimed at producing synthetic molecules that function as tumor suppressors downstream of p53.

Accordingly, presents the invention in one aspect as defined in the claims defines a substance which has the property to inhibit Cdk4, for use in one Method of medical treatment ready.

In In another aspect, the invention is as defined in the claims the use of a substance which has the property Cdk4 inhibit in the manufacture of a drug for treatment a hyperproliferative disorder ready.

In a preferred embodiment, it was found that the C-terminal portion of p21 ^WAF1 , which consisted of the peptide motif KRRLIFSK, completely inhibited cyclin Cdk4 activity and prevented the phosphorylation of pRb (see 6 ).

In In a further aspect, the present invention provides as in claims defines a substance that has the property cyclin-D to bind and / or inhibit Cdk4, for use in a medical Treatment procedure ready.

In In a further aspect, the present invention provides as in claims defines the use of a substance that has the property To bind cyclin-D and / or to inhibit Cdk4 in the preparation a drug for the treatment of hyperproliferative disorder.

Based on experimental evidence given below and showing residues involved in the binding of peptide 2 (residues 16-35) and the crystal structure available for p27 (related to p21), the following general formula is given Peptides useful in accordance with various aspects of the present invention are provided:
KxxRRyFzP
where x may be any amino acid, y and z may be hydrophobic and each of the underlined residues may be missing or different, ie may be another amino acid. Hydrophobic residues may be alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine. Any or both of the amino acids R may be replaced by other basic residues, especially lysine (K) or histidine (H).

In the present invention, "an active part" means a peptide smaller than the fragment of the p21 ^WAF1 amino ^acid sequence while still retaining the above-mentioned relevant property.

In the present invention, "functionally mimicking" means a substance which may not contain an active part of the p21 ^WAF1 amino ^acid sequence and is probably not a peptide at all, but which nevertheless has the above-mentioned relevant property.

In In the present invention, "a derivative" means a peptide obtained by varying its amino acid sequence was modified, for. B. by manipulation of the peptide encoding nucleic acid or by changing of the peptide itself. Such derivatives of the natural amino acid sequence can one Insertion, an addition, a deletion or a substitution of a or more amino acids without substantially increasing the essential activity of the peptides change. An example for a derivative is the p21WAF1 mutant in which D is at position 149 of the Full length protein has been replaced by A, said mutant having increased cyclin D1-Cdk4 inhibitory activity.

preferred Substances according to certain embodiments of the present invention do not bind and / or disrupt PCNA p21 interaction or binding with PCNA not.

Cell cycle arrest may be due to various aspects according to the present invention in Rb-negative and / or Rb-positive cells, such as in the below Experiments is shown.

The present invention can use compounds comprising any of the above-mentioned substances coupled to carrier molecules, allowing in vivo delivery of the compounds into cells. In one embodiment, the carrier molecule is a 16-aa peptide sequence derived from the homodomain of Antennapedia (eg, as sold under the name "Penetratin") which are linked through a terminal Cys residue to one of the above sequences can. Alternatively, as in the examples described below, a carrier peptide (having the sequence RQIKIWFQNRRMKWKK) can be synthesized so that it is directly linked to peptide fragments. The "penetratin" molecule and its properties are described in the WO 91/18981 described.

Therefore The present invention provides in various aspects for a fault or interrupting the interaction between p21 and cyclin D1 and / or Cdk4 using a suitable agent.

Such an agent can be capable be to block a bond between a body that is at amino acid residues which were identified herein as binding or interaction with cyclin D1 and / or Cdk4 and / or for it Meaning are.

The complete sequence of the protein p21 has been explained and is described in the WO 95/13375 . WO 93/12251 and WO 95/06415 which are incorporated herein by reference.

Such Agents can be identified by screening methods comprising determining whether a tested agent binding the protein p21 or a suitable fragment, derivative, analog or functional Mimetics thereof with cyclin D1 and / or Cdk4 or a relevant Fragment, derivative, analog or a functional mimetic thereof inhibits or interrupts.

suitable Fragments of p21 include those which have residues as identified herein include. Smaller fragments as well as derivatives, analogues and functional Mimetics of this fragment can to similar ones Be used manner, for. B. peptides using a Method, such. As alanine scanners were identified.

In another aspect of the invention, wherein experiments using the Use the peptides described herein, as well as the use of the Peptides in the context of modulating the interaction of p21 with Cyclin D1 and / or Cdk4, these peptides may also be described as p21 mimetics useful be the interaction of p21 and other cyclin-Cdk interactions, especially of G1 complexes such as cyclin E-Cdk2. The various embodiments of the invention described above and below with respect to cyclin D1 and / or Cdk4 is with the necessary modifications applicable to these other cyclins or Cdks.

screening process and tests are described in more detail below.

A Class of agents that can be used to bind To destroy p21 and cyclin D1 and / or cdk-4 are on sequence motifs p21-based peptides that interact with cyclin D1 and / or Cdk4. Such peptides tend to be small molecules, and may be about 40 or less amino acids be long, preferably they are about 35 or less amino acids long, even more preferably about 30 or less amino acids, more preferably about 25 or less amino acids long, more preferably about 20 or less amino acids long, more preferably about 15 or less amino acids in length, more preferably about 10 or less amino acids long or 9, 8, 7, 6, 5 or less amino acids long. The present The invention also encompasses peptides, the sequence variants or derivatives of a wild-type p21 sequence.

Preferably, the amino acid sequence has homology with a fragment of the p21 relevant fragment sequence, preferably having at least about 30% or 40% or 50% or 60% or 70% or 75% or 80% or 85% homology or at least with about 90% or 95% homology. A peptide fragment of p21 can therefore 1, 2, 3, 4, 5, more than 5 or more than 10 amino acid changes, such as. Substitutions with respect to the wild-type sequence.

One Derivative of a peptide, for the specific sequence disclosed herein can be understood in particular embodiments the same length how to have the specific peptide or be shorter than this. In other embodiments may be the peptide sequence or a variant thereof in a larger peptide, as described above, that is an additional one May or may not include section of p21. 1, 2, 3, 4 or 5 or more additional Amino acids, next to the relevant specific peptide fragment in p21, or heterologous can do this be included at one or both ends of the peptide.

As As is well known, homology is at the amino acid level generally in terms of amino acid similarity or identity. similarity allows a "conservative Variation ", i. Substitution of a hydrophobic residue, such as. Isoleucine, valine, Leucine or methionine, for another or substitution of one polar remainder for another, such as Arginine for Lysine, glutamic acid for aspartic acid or Glutamine for Asparagine. similarity can as the TBLASTN program Biol. 215, 403-10 (1990), which is in standard use is defined and defined in the field of the invention. The homology can be over the full length extend the relevant peptide or a related sequence of about 5, 10, 15, 20, 25, 30 or 35 amino acids compared to the relevant one Wild-type amino acid sequence.

As found, can various peptide sequences and peptides and non-peptide analogs and mimetics, as described below.

Various Aspects of the present invention use a substance that a single molecule or a composition containing two or more components which comprises a peptide fragment of p21 which has a as described above and / or elsewhere herein disclosed sequence comprises a peptide consisting essentially of such a sequence, a peptide that is a sequence variant, a derivative or analog Sequence, or a non-peptide analogue or a mimetic, which has the ability Cyclin D1 and / or Cdk4 bind and / or the interaction between p21 and cyclin D1 and / or Cdk4 to interrupt or disrupt.

variants include peptides in which individual amino acids are replaced by other amino acids can be that as known in the art and given above is closely related.

Non peptide-type Mimetics are further described below.

As is known, a peptide according to the present Invention and for use in various aspects of the present invention Invention comprise a fragment of p21 as disclosed or substantially consist of such. B. a fragment whose sequence is above cited becomes. If one or more additional Includes amino acids are, so can these amino acids be p21 or heterologous to p21 or foreign to p21 be. A peptide may also be included in a larger fusion protein be, especially in cases in which the peptide is linked to a non-p21 sequence (i.e., heterologous or foreign), such. A polypeptide or protein domain fused is.

The invention also uses derivatives of the peptides, including a peptide bound to a binding partner, e.g. As an effector molecule, a label, an active ingredient, a toxin and / or a carrier or transport molecule. Methods to bind the peptides of the invention to both peptidyl and nonpeptidyl binding partners are well known in the art. In one embodiment, the carrier molecule is a 16-aa peptide sequence derived from the homodomain of Antennapedia (eg, as sold under the name "Penetratin"), which can be coupled to a peptide through a terminal Cys residue. The "penetratin" molecule and its properties are described in the WO 91/18981 described.

Peptides can be produced in whole or in part by chemical synthesis. The compounds of the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are available over a large area (see, e.g., J. M. Stewart & JD Young, Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Company, Rockford, Illinois (1984), M. Bodanzsky & A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984), and Applied Biosystems 430A User's Manual, ABI Inc., Foster City, California), or by the liquid phase method or any combination of solid phase, liquid phase and solution chemistry, e.g. By first completing the respective section of peptide and then, if desired and appropriate, after removal of any protecting groups which may be present by introducing the radical X by reaction of the respective carbon acid or sulfonic acid or a reactive derivative thereof, in solution.

A another convenient way, a Peptidylmolekül (peptide or polypeptide) is produced by expression of the nucleic acid encoding it Use of nucleic acid in an expression system.

in the Generally, it will be nucleic acid as an isolate, in isolated and / or purified form or free from or substantially free of materials with which it of course is associated, such. B. free of or substantially free from nucleic acid flanking the gene in the human genome, with the exception of the possibility one or more regulatory sequence (s) for expression. nucleic acid may be wholly or partially synthetic and may include genomic DNA, include cDNA or RNA. In cases, in which nucleic acid RNA should include the reference to the sequence shown as a reference on the RNA equivalent be understood, with T replaced by U.

nucleic acid sequences, the for a polypeptide or peptide may be used by those skilled in the art the information and references contained herein as well as the methods known in the art (see e.g. Sambrook, Fritsch & Maniatis, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), and Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons (1992)) in view of the available nucleic acid sequence and clones are easily made. These methods include (i) the use of the polymerase chain reaction (PCR) to sample such nucleic acids, z. From genomic sources, (ii) chemical Synthesis or (iii) Preparation of cDNA sequences. For p21 fragments Coding DNA can be prepared in any suitable manner known to those skilled in the art is to be produced and used, inter alia, by selecting coding DNA, identification of suitable restriction enzyme recognition sites on both sides of the section to be expressed, and Cut out the said section from the DNA. The section can then be operably linked to a suitable promoter in a commercial available Be bound to standard expression system. Another recombination approach is the amplification of the relevant part of the DNA with appropriate PCR primers. Modifications of these p21 sequences can, for. Example, by means of site-directed mutagenesis, carried out to express of modified p21 peptide or codon preference in the host cells used to express the nucleic acid, to consider.

Around the expression of the nucleic acid sequences to reach the sequences into a vector having one or more control sequences, to the nucleic acid operably linked to be incorporated into their expression Taxes. The vectors can other sequences, such as. Promoters or enhancers, to the expression of the inserted nucleic acid, nucleic acid sequences to propel the polypeptide or peptide as a fusion and / or nucleic acid, the for secretion signals coded, so that produced in the host cell Polypeptide is secreted by the cell. The polypeptide can then pass through Transform the vectors into host cells containing the vector is functional, culturing the host cells, so that the polypeptide and recovering the polypeptide from the host cells or the surrounding medium can be obtained. For this purpose be on the Area of the invention uses prokaryotic and eukaryotic cells, among others other tribes E. coli, yeast and eukaryotic cells such as COS or CHO cells.

One Process for the preparation of a polypeptide or peptide (as disclosed) includes the expression of nucleic acid encoding the polypeptide or peptide encoded (generally nucleic acid according to the invention). This can be done easily by growing a host cell in culture containing such a vector under suitable conditions, which lead to or enable the expression of the polypeptide, be achieved. Polypeptides and peptides can also be used in in vitro systems, such as As reticulocyte lysate can be expressed.

systems for cloning and expression of a polypeptide in a variety of different ways Host cells are well known. Suitable host cells include bacteria, eukaryotic cells, such. B. mammalian or yeast cells, and Baculovirus. Mammalian cell lines those in the field of the invention for the expression of a heterologous Polypeptides available include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others. A common, preferred bacterial host is E. coli.

Suitable vectors can be selected or constructed containing the appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as appropriate. Vectors may be plasmids, viral, e.g. As "phage" or phagemid, depending on their suitability. For further details see z. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Srping Harbor Laboratory Press (1989). Many known methods and procedures for manipulating nucleic acid, e.g. For the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells len and gene expression and analysis of proteins are described in Ausubel et al. (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons (1992).

The nucleic acid can be integrated into the genome (eg chromosome) of the host cell. The integration can be done in accordance with standard procedures by inclusion of sequences promoting recombination with the genome. The nucleic acid may be located on an extra-chromosomal vector in the cell or otherwise identifiable heterologous with the cell or be alien to this.

The introduction the nucleic acid in a host cell, the (in particular for an in vitro introduction) in general without restriction be referred to as "transformation" can, by any available Procedure done. For eukaryotic cells can suitable methods calcium phosphate transfection, DEAE-dextran, Electroporation, liposome-mediated transfection and transduction using retroviruses or other viruses, e.g. B. vaccinia or for Insect cells, baculovirus. For bacterial cells may be appropriate Procedures Calcium chloride transformation, electroporation and transfection by bacteriophages. As an alternative, direct Injection of the nucleic acid be used.

Marker genes, such as B. antibiotic resistance or sensitivity genes, can be used to identify clones encoding the nucleic acid of As well known in the art is.

On the introduction can be causing or enabling expression from the nucleic acid follow, z. By culturing host cells (which include cells can, actually were transformed, although it is more likely that the cells Progeny of the transformed cells are), under the conditions for the Expression of the gene such that the encoded polypeptide (or peptide) is produced. When the polypeptide is attached to a suitable signal leader peptide coupled expressed, it may be from the cell into the culture medium be secreted. After the production by expression can one Polypeptide or peptide, depending on the particular situation, from the Host cell and / or the culture medium isolated and / or purified and then as desired be used, for. In the formulation of a composition, one or more additional Components, such. B. a pharmaceutical composition, the one or more pharmaceutically acceptable excipients, vehicles or carrier (see, for example, below).

The introduction a nucleic acid, the one for one Peptidylmolekül according to the present Invention can take place in vivo by means of gene therapy, to the interaction between p21 and cyclin D1 and / or ckd4 too interrupt or disturb.

Therefore For example, a host cell containing the nucleic acid, e.g. B. as a result of introduction of nucleic acid into the cell or an ancestor of the cell and / or genetic modification the sequence leading to the cell or the ancestor (with the introduction or modification performed in vivo or ex vivo is endogenous, contained in an organism (eg in soma) being an animal, especially a mammal, human or non-human can be, such as A rabbit, a pig, a rat, a mouse or other rodent, a cat, a dog, a pig, a sheep, a goat, a cattle or horse or a bird, such as A chicken.

behind it can be a therapeutic goal. (Gene therapy will be below Also, the presence of a mutant, allelic, derivative sequence or sequence variant in the cells of an organism, in particular if this is in the place of a homologous endogenous sequence, a use of the organism as a model for testing and / or Studying substances allow which the activity modulate the encoded polypeptide in vitro or otherwise from therapeutic potential. Conveniently, you can Tests for however, such substances in vitro, in host cells or in cell-free Systems performed become.

suitable Screening methods are conventionally used in the art used. They include methods such. Radioimmunoassay, scintillation proximity test and ELISA methods. Suitably either the p21 protein or fragment or cyclin D1 and / or Cdk 4 or fragment or an analog, derivative, variant or functional mimetic thereof immobilized, after which the other in the presence of the agents to be tested is applied. In a scintillation proximity test is a biotinylated protein fragment streptavidin-coated, beads impregnated with a scintillant (produced from Amersham). The binding of radioactively labeled peptide is then determined by determination of those induced by radioactivity Scintillation measured as the radioactive peptide immobilized to the Fragment binds. Agents that intercept this process are therefore Inhibitors of interaction.

In a general aspect, the present invention provides a test method as described in An for a substance with the ability to disrupt the interaction or binding between p21 and cyclin D1 and / or Cdk4.

Therefore can be a test compound showing the binding or interaction between said substances (eg comprising a p21 fragment and a Cyclin D1 and / or Cdk-4 fragment) interrupts, reduces, interferes or wholly or partially and which can modulate Cdk4 activity become.

One another general aspect of the present invention ceases Test method as in the claims defined for a substance ready, which, depending on the situation, is capable of the relevant region to bind by p21.

A Test compound found to be relevant Can bind to its ability to be tested p21 interaction or binding with cyclin D1 and / or Cdk 4, and / or on the ability the Cdk4 activity or other activities, which are mediated by p21, as described above, too influence.

The execution a test method according to the present Invention may be of isolation and / or manufacture and / or Using a compound, substance or molecule followed which (s) are the ability to interfere with the interaction between p21 and cyclin D1 and / or Cdk4 and / or the p21-mediated Cdk4 activity to inhibit was tested positive.

The exact format of a test of the invention may be varied by one skilled in the art through routine skill and knowledge. The interaction between substances may, for. In vitro by labeling one with a detectable label and contacting it with the other immobilized on a support material. Suitable detectable labels, particularly for peptidyl substances, include ³⁵ S-methionine, which can be incorporated into recombinantly produced peptides and polypeptides. Recombinantly produced peptides and polypeptides can also be expressed as a fusion protein containing an epitope which can be labeled with an antibody.

The on a solid carrier immobilized protein can be detected using an antibody against immobilized this protein, which bound to a solid support is, or using other methods known per se are. A preferred in vitro interaction may be a fusion protein using glutathione-S-transferase (GST). This can immobilized on glutathione agarose beads. In an in vitro test format of the type described above, a test compound can be determined by determination their ability the amount of labeled peptide or polypeptide attached to the immobilized GST fusion polypeptide binds to be minimized. That can determined by fractionation of the glutathione-agarose beads by SDS-polyacrylamide gel electrophoresis become. Alternatively, you can the beads rinsed be used to remove bound proteins, and the amount of protein which has been tied can by counting the amount of markers present, e.g. B. in a suitable scintillation be determined.

One Test according to the present The invention may also take the form of an in vivo test. The in vivo test can in a cell line, such. A yeast strain or a mammalian cell line, in which the relevant polypeptides or peptides of one or more Vectors that have been introduced into the cell are expressed, carried out become.

The ability a test compound, the interaction or binding between p21 and cyclin D1 and / or Cdk4 may be disrupted by use of a so-called two-hybrid test.

A polypeptide or peptide, e.g. B., which, depending on the situation, a fragment of p21 or cyclin D1 / Cdk4 contains, or a peptidyl analog or a variant thereof, as disclosed, can be attached to a DNA-binding domain such. B. the yeast transcription factor GAL 4, fused. The GAL-4 transcription factor comprises two functional domains. These domains are the DNA-binding domain (GAL4DBD) and the GAL4 transcriptional activation domain (GAL4TAD). By fusing a polypeptide or peptide to one of these domains and another polypeptide or peptide to its respective counterpart, a functional GAL-4 transcription factor is restored only when two polypeptides or peptides of interest interact. Therefore, the interaction of the polypeptides or peptides can be measured by the use of a reporter gene, likely bound to a GAL-4 DNA binding site, which is capable of activating transcription of the reporter gene. This assay format is described by Fields & Song, Nature 340, 245-246 (1989). This type of test format can be used both in mammalian cells and in yeast. Other combinations of DNA-binding domains and transcriptional activation domains are available in the art and may be preferred, such as e.g. The LexA DNA binding domain and the VP60 transcriptional activation do activator domain.

Around z. For example, for purposes of illustration, a Lex / VP60 two-hybrid screen yeasts can or mammalian cells with a reporter gene construct containing a selective marker protein (e.g. For β-galactosidase or luciferase encoding) are expressed, transformed. Of the Promoter of this gene has been created to be a binding site for the LexA DNA binding protein contains. Gene expression from this plasmid is usually very low. Two more expression vectors can be transformed into the yeast which contains the selectable marker expression plasmid, wherein one the coding sequence for the full-length LexA gene contains which is bound to a multiple cloning site. This multiple Cloning site is used to clone a gene of interest d. H. For a p21 or cyclin D1 / Cdk4 polypeptide or peptide according to the present invention Invention encoding in-frame on the LexA coding region. The second expression vector then comprises the activation domain of the herpes simplex transactivator VP16 linked to a test peptide sequence or more preferably a library at sequences, which for Peptides with different, e.g. Random, encoding sequences, was merged. The two plasmids simplify expression from the reporter construct containing the selectable marker only when the LexA fusion construct with a polypeptide or Peptide sequence derived from the peptide library interacts.

On looking for peptides or other substances affecting the interaction between a p21 polypeptide or peptide and a cyclin D1 / Cdk-4 polypeptide or peptide disrupt, a modification thereof uses the p21 or cyclin D1 / Cdk4 polypeptide or peptide as a fusion with the LexA DNA binding domain and its counterpart cyclin D1 / Cdk4 or p21 polypeptide or peptide as a fusion with VP60 and a third expression cassette based on a separate expression vector from which a peptide or a peptide library of different and / or random Sequences can be expressed. A reduction in reporter gene expression (For example, in the case of β-galactosidase a weakening the blue color) is due to the presence of a peptide that the p21 / cyclinD1 and / or Cdk4 interaction interrupts, wherein this interaction for the transcriptional activation of the β-galactosidase gene is required. If a test substance is not peptidyl and not of the coding nucleic acid in a third expression cassette can be expressed, so can be a similar one System in which the test substance administered exogenously becomes.

As cited can instead of LexA and VP60 other similar combinations of proteins used together, a functional transcriptional activator form, such. The GAL4 DNA binding domain and the GAL4 transcriptional activation domain.

Becomes conducted a two-hybrid test, to look for substances that affect the interaction between two Interfere with polypeptides or peptides, so may the use of mammalian cells instead of yeast cells are preferred. The same apply Principles, and suitable methods are well known to those skilled in the art.

The Amount of the test substance or compound that results in a test of the invention to be added can, normally, be made dependent on Type of compound used, using trial-and-error experiments certainly. Typically, you can Concentrations of suspected Inhibitor compounds of about 0.01 to 100 nM are used, z. From 0.1 to 10 nM. Larger concentrations can used when a peptide is the test substance.

Links, which can be used can natural or synthetic chemical compounds used in drug screening programs be used. Plant extracts that characterized several or uncharacterized components may also be used.

Antibodies that in both proteins directed to site of interaction are another class of putative inhibitor compounds. Candidate inhibitor antibodies can be characterized and their binding regions are detected to be single-chain antibodies and To provide fragments thereof for interrupting the interaction are responsible.

Antibodies can be under Application of standard methods known in the art to be obtained. Methods for the production of antibodies include the immunization of a mammal (eg mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies can be from immunized animals using any of a number in the field of the invention known methods are obtained, and preferably using a binding of the antibody to the antigen of interest, to be screened. It can z. B. Western blotting method or immunoprecipitation are used (Armitage et al., Nature 357, 80-82 (1992)). Isolation of antibodies and / or antibody-producing Cells from an animal may be accompanied by the step of killing the animal.

As an alternative or supplement to immunizing a mammal with a peptide, antibody specific for a protein may be prepared from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. Using λ-bacteriophages or filamentous bacteriophages having functional immunoglobulin binding domains on their surfaces; see, eg. B. WO 92/01047 , The library may be naïve, that is, constructed from sequences obtained from an organism that has not been immunized with any of the proteins (or fragments), or may be one generated using sequences from an organism encoding the antigen of Interest was exposed was constructed.

Antibodies according to the invention can be modified in many ways. In fact, the term "antibody" should include any binding substance a binding domain with the required specificity cover and be understood as such. Therefore, the invention covers Antibody fragments, Derivatives, functional equivalents and homologues of antibodies containing synthetic molecules and molecules, whose shape mimics that of the antibody, what enables them to bind an antigen or epitope.

Examples for antibody fragments, who are capable to bind an antigen or another binding partner are Fab fragment consisting of VL, VH, CI and CH1 domains; the Fd fragment consisting of the VH and CH1 domains; the Fv fragment, that from the VL and VH domains a single arm of an antibody consists; the dAb fragment consisting of a VH domain; isolated CDR regions and F (ab ') 2 fragments, a bivalent fragment that includes two Fab fragments that span over a disulphide bridge are bound to the hinge region. Single chain Fv fragments are as well included.

A hybridoma producing a monoclonal antibody according to the present invention may be subjected to a genetic mutation or other changes. Further, it will be appreciated by those skilled in the art that a monoclonal antibody may be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such methods may include introducing DNA encoding the variable immunoglobulin region or complementarity determining regions (CDRs) of one antibody into the constant regions or constant regions plus framework regions of another immunoglobulin. See, for example, B. EP 184187 A . GB 2188638 A or EP-A-0239400 , The cloning and expression of chimeric antibodies is described in EP-A-0120694 and EP-A-0125023 described.

hybridomas the capable are, antibodies with the desired Binding properties are within the scope of present invention, as well as host cells, eukaryotic or prokaryotic, the nucleic acid which are available for antibodies (including Antibody fragments) encoded, and are capable of their expression. The invention provides also provide methods of producing the antibodies, comprising Breed a cell that is capable is the antibody to produce under conditions in which the antibody produces and preferably secreted.

The Reactivity of antibodies in a sample can be determined by any suitable method. Labeling with single reporter molecules is one possibility. The reporter molecules can be direct or generate indirectly detectable and preferably measurable signals. The binding of reporter molecules can be directly or indirectly, covalently, z. B. via a peptide bond, or not to be covalent. A bond over a peptide bond may be the result of recombinant expression of a Be a gene fusion for the antibody and the reporter molecule coded.

A preferred species is by covalent attachment of each antibody a single fluorescent dye, phosphorus or laser dye with spectrally isolated absorption or emission properties. Suitable fluorescent dyes include fluorescein, rhodamine, Phycoerythrin and Texas Red. Suitable chromogenic dyes include Diaminobenzidine.

Other Reporters include macromolecular colloidal particles or particulate material, such as B. colored Latex beads, magnetic or paramagnetic, as well as biological or chemically active agents that, directly or indirectly, are detectable Can cause signals, which is visibly observed, electronically detected or otherwise Way can be included. These molecules can be enzymes that catalyze reactions that develop colors or change or z. B. changes in electrical properties. They can be molecularly excitable, so that electronic transitions between energy states lead to characteristic spectral absorptions or emissions. she can Chemical entities that are used in conjunction with biosensors be used. Biotin / avidin or biotin / streptavidin and alkaline phosphatase detection systems can be used.

The mode of detection of binding is not a feature of the present invention, and One skilled in the art will be able to choose a suitable mode according to his preferences and general knowledge.

Antibodies can also used to prepare a polypeptide or peptide according to the present invention Invention to clean and / or to isolate, for. B. after production of the polypeptide or peptide by expression coding therefrom Nucleic acid. antibody can in a therapeutic context (which may include prophylaxis) useful be the p21 / cyclin D1 / Cdk4 interaction in terms of a Inhibition of Cdk4 activity and thus to interrupt cell proliferation. Antibodies may, for. B. are microinjected into cells, such as at a tumor site.

Other Candidate inhibitor compounds can be used to model the 3-dimensional structure of a Polypeptide or peptide fragments based as well as on the use from rational drug design to potential inhibitor compounds with special properties of molecular shape, size and charge provide.

A Compound that was found to have the ability has the Cdk4 activity too have therapeutic potential in antitumor treatment and may be used in combination with other antitumor compounds become. In such a case, the test of the invention, if he performed in vivo will not measure the degree of inhibition of binding or modulation of Cdk4 activity, which is caused by the tested compound. Instead may have an effect on tumorigenicity and / or cell viability be measured. It may be that such a modified test in parallel is performed on or after the main test of the invention, to confirm, that any effect on tumorigenicity or and / and cell viability the result of inhibition of binding or interaction between p21 and cyclin D1 / Cdk4 caused by the inhibitor compound and not merely a general toxic effect.

To the identification of a substance or agent that contains the Cdk4 activity modulated or influenced, the substance or agent may continue to be examined. Furthermore, it can be manufactured and / or in the production, d. H. in the preparation or formulation, a composition such as a medicament, a pharmaceutical composition or an active ingredient become. these can Be administered to individuals.

As has been highlighted, the agent may be a Peptidyl, z. A peptide, which contains a sequence as stated above, or a functional analog of such a peptide.

As As used herein, the term "functional analog" refers to peptide variants or organic compounds having the same functional activity as the said peptides that can interfere with the binding between p21 and cyclin D1 / Cdk4. Examples of such analogs include chemical compounds, so are modeled to the three-dimensional structure of the p21 or cyclin D1 / Cdk4 domain in the contact area and more particularly, the arrangement of the key amino acid residues shown above are identified, as they occur in human p21 to resemble.

In In a further aspect, the present invention provides, as in claims defines the use of the above substances in procedures to design or screen for mimetics of these substances.

• (i) Analyse einer Substanz mit der biologischen Aktivität, um die Aminosäurereste zu bestimmen, die für die Aktivität, ein Pharmakophor zu bestimmen, essentiell und wichtig sind, und
• (ii) Modellierung des Pharmakophors, um Kandidaten-Mimetika mit der biologischen Aktivität zu kreieren und/oder zu screenen.

A method of designing mimetics of p21 ^WAF1 having the biological activity of binding or inhibiting Cdk4, allosterically inhibiting the activity of Cdk4, and / or the activity of binding cyclin D1 may comprise:

• (i) analyzing a substance having the biological activity to determine the amino acid residues that are essential and important for the activity of determining a pharmacophore, and
• (ii) modeling the pharmacophore to create and / or screen candidate mimetics with biological activity.

suitable Modeling methods are known in the art. This includes the design of the so-called "mimetics", which is the study of functional Interactions between fluorogenic oligonucleotides and the molecules and the design of compounds containing functional groups that are arranged in such a way that they are Can reproduce interactions, involved.

The Designing mimetics of a known pharmaceutically active compound is a well-known approach to the development of pharmaceutical products, which are based on a lead connection. That may be desirable in cases in which the active compound is difficult or expensive to synthesize is or where they are for a certain mode of administration is not suitable, for. B. are suitable peptides do not work well as agents for oral compositions, as they tend to rapidly become proteases in the digestive tract to be mined. Mimicking design, synthesis and testing can be used be a random screening of large numbers of molecules on a target property to avoid.

There are some steps that are usually in the Design of a mimetic of a compound with a said goal property are performed. First, the particular parts of the connection which are critical and / or important for the determination of the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. By sequentially substituting each of the radicals. These parts or residues, which are the active region of the compound, are known as their "pharmacophore".

is once the pharmacophore is found, its structure will be commensurate modeled on its physical properties, e.g. B. stereochemistry, Binding, size and / or Charge, using data from a number of sources, z. B. Spectroscopy method, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which the charge and / or volume of a pharmacophore instead of the Bond between the atoms modeled) and other methods can be used in this Modeling process can be used.

In A variant of this approach is the three-dimensional structure of the ligand and its binding partner. This can be especially useful when the ligand and / or binding partner is the conformation of the bond change, to allow the model to to consider this design of the mimetic.

One template molecule is then selected on grafted chemical groups that mimic the pharmacophore can be. The template molecule and the grafted-on chemical groups can easily selected so that the mimetic is easy to synthesize is pharmacologically acceptable and is not degraded in vivo and while maintaining the biological activity of the lead compound. The Mimetic or the mimetics found by this approach was (can), can / can then be screened to see if they have the target feature or to what extent they have this. Further optimization or modification can then carried out to one or more final mimetics for in vivo or clinical testing receive.

The Mimetic or the mimetics found by this approach was (can), can / can then be screened to see if they have the target feature or to what extent they have this. Further optimization or modification can then carried out be one or more final mimetics for in vivo or clinical testing to obtain.

Furthermore, provides the present invention as defined in the claims, the use of a peptide or a derivative, an active one Section, analogues or a variant of it ready, the / that is able to screen for a substance that is capable is to bind Cdk4 and / or to inhibit Cdk4 activity, to bind Cdk4 and / or the Cdk4 activity to inhibit.

Usually becomes an inhibitor in an isolated and / or purified form, d. H. essentially pure, provided. That may be the presence in a composition in which it accounts for at least about 90% of the active ingredient, more preferably at least about 95%, more preferably at least about 98%. Such a composition, however, can inert carrier materials or other pharmaceutically and physiologically acceptable excipients. As noted below, a composition in addition to an inhibitor compound as disclosed, one or more others molecules therapeutic use, such as. An antitumor agent.

A A substance which, in accordance with the disclosures contained herein, as a modulator of the p21 and cyclin D1 / Cdk4 interaction and / or the Cdk4-mediated RB phosphorylation or other substrates of Cdk4 or others p21-mediated activity, Property or metabolic pathway has been identified in one pharmaceutical composition, drug, drug or another composition comprising such a substance, a method involving the administration of such a composition to a patient, e.g. As antitumor or other antiproliferative Treatment, the preventive treatment include can, a use of such a substance in the preparation of a composition for administration, e.g. As antitumor or other antiproliferative Treatment, and a method for producing a pharmaceutical Composition, which adds the addition of such a substance a pharmaceutically acceptable excipient, vehicle or carrier as well may contain other ingredients.

A Substance, such as As an inhibitor of p21 and cyclin D1 and / or Cdk4 interaction or Binding, may be for use in a method of treatment of human or animal body by a therapy involving Cdk4 activity or other p21-mediated activity in cells, e.g. As tumor cells, influenced.

Thus, a method of modulating Cdk4 activity or other p21-mediated activity in a cell comprises administering a An agent that inhibits or blocks the binding of p21 to cyclin D1 and / or Cdk4 protein, such a method in the treatment of cancer or other diseases or disorders, including malignancies, in which the inhibition of cellular growth and / or proliferation is desired, is useful.

One Methods of treating tumors include administering a Agent to a patient, the binding of p21 to cyclin D1 and / or Cdk4 bothers.

No matter whether it is a polypeptide, an antibody, a peptide, a nucleic acid molecule, or a small molecule Molecule, is a mimetic or other pharmaceutically useful compound, which is to be administered to an individual, the administration is preferably in a "prophylactic effective amount "or a "therapeutically effective Quantity "(depending on Situation, although prophylaxis can be considered as a therapy) this is sufficient to show positive effects on the individual. Actually amount administered, the frequency and the schedule of administration depend on the type and severity of the disease to be treated. The prescription of treatments, z. B. decisions about the dosage etc., is the responsibility of the general practitioners and other medical specialists.

A Composition may be alone or in combination with other treatments be administered, either simultaneously or sequentially, as appropriate the disease to be treated.

pharmaceutical Compositions for use according to the present invention can, additionally to the active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such Materials should not be toxic and should not affect the efficacy of the product Do not disturb the active substance. The exact nature of the vehicle or other material on the mode of administration, which can be administered orally or by injection, z. Cutaneous, subcutaneous or intravenous.

pharmaceutical Compositions for oral administration may take the form of tablets, Capsules, powders or liquids to have. A tablet may be a solid carrier, such as. Gelatin or an adjuvant. liquid Pharmaceutical compositions generally include one liquid Carrier, such as As water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. physiological Saline, Dextrose or other saccharide solutions or glycols, such as. As ethylene glycol, propylene glycol or polyethylene glycol, can to be included.

to intravenous, cutaneous or subcutaneous injection or for injection at the site Of the complaints, the active ingredient has the form of a parenteral acceptable aqueous Solution, which is pyrogen-free and has a suitable pH, a suitable Isotonia and stability having. The average educated expert is easily in capable of finding suitable solutions to produce, for. Isotonic vehicles, such as sodium chloride injection, Ringer injection, cycled Ringer injection. Preservatives, Stabilizers, buffers, antioxidants and / or other additives can, as needed, includes.

Examples for above mentioned standing Procedures and procedures are in Remington's Pharmaceutical Sciences, 16th Edition, A. Oslo (ed.) (1980).

The Agent may be local to a tumor site or to another desired site be administered or it may be administered in a manner targeting tumor or other cells as a target.

Targeting therapies can used to make the drug more specific by use of targeting systems, such as B. antibody or cell specific Ligands to supply specific cell types. Targeting may be due a number of reasons he wishes be, z. If the agent is unacceptably toxic or if it is otherwise it would require too high a dose or otherwise would be capable of Invade target cells.

Instead of To administer these agents directly, they can in the target cells by expression from a coding gene introduced into the cells, z. In a viral vector (a variant of the VDEPT method - see below). The vector may be specific to, too targeting cells or regulatory elements included, more or less selectively turned on by the target cells become.

The agent can be administered in a precursor form for conversion to the active form by an activating agent that is produced in or targeted by the cells to be treated. This type of approach is sometimes referred to as ADEPT or VDEPT, wherein the former involves targeting the activating agent to the cells by conjugation to a cell-specific antibody, while the latter controls the production of the activating agent, e.g. An enzyme, in a vector by expression from encoding DNA in a viral vector (see eg. EP-A-415731 and WO 90/07936 ).

A composition may be alone or in Combination with other treatments, either simultaneously or sequentially, depending on the condition being treated, e.g. Cancer, viral infections or any other condition in which a p21-mediated effect is desired.

Nucleic acid, the for a Polypeptide or peptide capable of the p21 and cyclin D1 and / or Disrupting Cdk4 interaction or binding and / or the Cdk4 activity or other p21-mediated cellular To induce or modulate metabolic pathways or functions, can be used in gene therapy procedures, e.g. B. in the treatment of individuals aiming at a tumor (in whole or in part) prevent or cure, eg. In cancer or other disorders, a loss of proper regulation of the cell cycle and / or cell proliferation, as well as other disorders in which a specific Cell death desirable is, such. In certain viral infections.

vectors such as As viral vectors, have been used in the prior art, to nucleic acid in a big one To introduce selection of different target cells. Typically the vectors opposite Target cells exposed, so that the transfection in a sufficient Part of the cells can take place to be a useful therapeutic or Prophylactic effect from the expression of the desired To provide polypeptide. The transfected nucleic acid can permanently into the genome of each of the targeted tumor cells be incorporated and thus provide a long-lasting effect, Alternatively, the treatment may also be repeated periodically.

A number of vectors, both viral vectors and plasmid vectors, are known in the art. See U.S. Patent No. 5,252,479 and WO 93/07282 , Specifically, a number of viruses have been used as gene transfector vectors, including papovaviruses such as SV40, vaccinia viruses, herpesviruses including HSV and EBV, and retroviruses. Many gene therapy protocols in the art have used crippled murine retroviruses.

When Alternative to using viral vectors include other known ones Method of insertion of nucleic acid in cells electroporation, calcium phosphate co-precipitation, mechanical processes, such as Microinjection, liposome-mediated transfer and direct DNA uptake and receptor-mediated DNA transfer.

receptor-mediated Gene transfer in which the nucleic acid via polylysine bound to a protein ligand, wherein the ligand specific for one on the surface is the target cell receptor is an example of a method for specific targeting of nucleic acid to specific cells.

One Polypeptide, peptide or other substance capable of the interaction of the relevant polypeptide, peptide or other, a substance as disclosed herein, or a nucleic acid molecule encoding a peptidyl coded, can be used in a set, eg. B. sealed in a suitable Container, which protects its contents from the external environment provided become. Such a kit may include instructions for use.

Various further aspects and embodiments The present invention will be apparent to those skilled in the art to the present disclosures. Some aspects and embodiments The invention will now be illustrated by way of example and reference on the following figures:

1 : The ability of peptides of p21 ^{WAF1 to interact} with Cdk4 and cyclin D1.

1a : a list of peptides 1-11 based on the sequence of p21 ^WAF1 . 1b : The p21 ^WAF1 peptides were bound to streptavidin ^agarose beads and added to reticulocyte lysates containing either Cdk4 or cyclin D1 labeled with [ ³⁵ S] -methionine. After extensive washing, bound proteins were analyzed using SDS-PAGE, followed by autoradiography. The bands were quantified using a bio-imager and whole-band analysis software (Millipore). The results are representative of 3 such experiments. "x" indicates beads without peptide.

2 : Addition of p21 ^WAF1 Based Peptides to Cyclin D1 Cdk4 Phosphorylation Assays.

Cyclin D1-Cdk4 assays were performed in vitro using lysates from an Sf9 insect cell followed by co-infection with Cdk4 and cyclin D1 baculovirus constructs and GST-Rb as substrate. p21 ^WAF1 peptides were added to the tests at a concentration of 17 μM, and the effect on Cdk4 activity was determined by SDS-PAGE and autoradiography. The drawing shows the quantification of the autoradiogram using biosensors, the relative binding is represented by the Cdk4 activity in the absence of the peptide. The data are representative of 4 experiments. "x" shows no addition of peptide.

3 : Quantification of peptide inhibition.

Peptides 4, 8, 2 and 10 were added to cyclin D1-Cdk4 assays at various concentrations between 0.01-34 μM. The drawing shows a plot of activity (%) relative to Cdk4 activity measured in the absence of the peptides, peptide concentration and I _0.5 for each peptide. The data show the mean of 3 experiments.

4 : Peptides 2 or 10 are not substrates for cyclin D1-Cdk4.

The Fig. 1 shows the results of the phosphorylation tests using peptides 2, 4 and 10.

5 : Size scan of peptide 10.

The Figure 1 shows the sequences of a series of peptides based on peptide 10 are based and designed to be the least inhibitory domain to find. The framed residues represent the minimal inhibitory domain The peptides were added to cyclin D1-Cdk4 assays and SDS-PAGE and autoradiography analyzed.

6 : Alanine Scan Mutations of Peptide 10.

Around Leftovers for The inhibition of Cdk4 by peptide 10 was crucial a series of point mutations was constructed in each residue was sequentially changed to alanine. The peptides were to cyclin D1 Cdk4 assays added and the results were analyzed by SDS-PAGE and autoradiography and then quantified using a bio-imager. The results are relative to Cdk4 activity in Absence of peptide expressed and represent 3 experiments. After the inventors identified the crucial residues they synthesized an unlabeled eight-amino acid peptide which containing R, L and F (KRRLIFS) and determined phosphorylation of GST-Rb by cyclin D1-Cdk4 in the presence of increasing concentrations this truncated peptide.

7 : Comparison of inhibitory peptides with full-length p21 ^WAF1 protein.

This shows the concentration curves for peptide 10, D to A mutant peptide 10, a peptide derived from p16INK4 (Fahraeus et al. (1996)) and full length his-p21 ^WAF1 , determined using the cyclin D1-Cdk4 assay , analyzed by SDS-PAGE, autoradiography and bioimaging, and the I _{0.5 of} each inhibitor. The results are the mean of 3 such experiments.

8th : Binding and Inhibitory Domains of p21 ^WAF1 . The hatched residues show the regions of p21 ^{WAF1 identified} in this study as being important for cyclin D1 and Cdk4 binding and Cdk4 inhibition in the N-terminal domain, as well as a new inhibitory domain in the C-terminus of p21 ^WAF1 , The residues found to be significant for the interaction of p21 ^WAF1 with PCNA (Warbrick et al., 1995) are shown in black. In addition, the smallest part of p21 ^WAF1 , which was found to ^inhibit CDK activity in vitro prior to the present study (Luo et al., 1995), is shown.

9 : Introduction of p21 ^WAF1 based peptides into cells.

A series of synthetic peptides based on the sequence of peptide 10 (peptides I, II and III, shown in FIG 9a ) were synthesized with a carrier peptide (hatched sequences). The underlined residue in peptide I is the M to A mutation which prevents PCNA binding. The peptides were added to proliferating HaCaT cells grown in DMEM plus 10% FCS. The cells were incubated for 24 hours, while pulse-labeled with 15 μM BrdU, fixed and then analyzed by FACS. The G ₁ , S and G ₂ phase distributions for untreated cells, peptide I at 25 μM, peptide II at 50 μM and peptide III at 25 μM were determined. 9b Figure 14 shows data represented as the% of cells in each phase compared to the total number of cells counted. The results of similar experiments using MCF7 and MRC5 cells are presented in 9c which shows the percentage of cells in each phase of the cell cycle in the presence and absence of peptide-I.

In a separate experiment was DMEM + 10% FCS alone or DMEM + 10% FCS containing either 25 μM Peptide I or 50 μM Peptide II was added to the HaCaT cells, which then lasted for 72 hours were starving. Samples were taken at the indicated times and analyzed by SDS-PAGE / Western blot stained for pRb. pRb represents hypophosphorylated Rb protein, and pRb * refers to hyperphosphorylated Rb protein. It should be noted that equal amounts of total protein per track and it appears that the antibody is preferentially recognizes phosphorylated forms of the Rb protein.

10 : Inhibition of Cyclin D1-Cdk4 Activity Using Derivatives of Peptide 2.

The figure shows the degree of inhibition of cyclin D1-Cdk4 activity using pRb as substrate by peptide-2- (2 in the Figure) and alanine-scan mutations of the peptide (with each residue is mutated sequentially to alanine). The activity is indicated relative to uninhibited activity. (No stands for no added peptides.) Peptides were present at a concentration of 10 μM. A similar pattern can be seen when peptide 2 mutants are bound to cyclin D1 expressed in reticulocyte lysates.

Important Leftovers for the binding and inhibition are the two arginine residues (R) and Phenylalanine (F), with lysine (K) and proline (P) also playing a role. This is different compared to residues responsible for the interaction of full-length protein with cyclin D1 were identified as crucial as these studies identify the LFG motif as the most important activity.

Experimental procedures

peptides

All Peptides were obtained from Chiron Mimotopes, Peptide Systems (Clayton, Australia). synthesized. Each peptide has a biotin-SGSG spacer at the C-terminus and a free N-terminus. The peptides were added in DMSO about 5 mg / ml dissolved, and then the inventors precisely determined their concentration by amino acid analysis (Smythe et al., (1988)). additionally the purity of the peptides was estimated by mass spectrometry. positive Ion electrospray mass spectrometry was on a triple quadrupole mass spectrometer (V.G. Quattro) in 50/50 / 0.1 water / acetonitrile / formic acid.

proteins

Cycline and CDKs-Cdk4 and cyclin D1, Cdk2 and cyclin E and Cdc2 and cyclin B were co-expressed in Sf9 insect cells infected with the appropriate baculovirus constructs. The cells were harvested by low-speed centrifugation two days after infection, and the pellet was added in an equal amount of 10 mM Hepes, pH 7.4, containing: 10 mM NaCl, 1 mM EDTA, and 0.1 mM phenylmethanesulfonyl fluoride, 2 mM DTT , lysed and centrifuged at 14,000 × g for 15 minutes. The supernatant was removed, aliquoted and immediately frozen in liquid nitrogen. Thawed lysate was used only once and was never frozen again. Labeled Cdk4 and cyclin D1 were produced by translation in the presence of [ ³⁵ S] methionine using an in vitro rabbit reticulocyte lysate translation kit (Promega).

His-tagged p21 ^WAF1 - Human p21 ^WAF1 was expressed in E. coli using a PET expression vector. The soluble p21 ^WAF1 protein ^{fraction was purified} using a nickel ^{chelate column} and following the manufacturer's instructions (Pharmacia). The eluted protein peak was dialyzed against 25 mM Hepes, pH 7.4 containing: 0.1 mM EDTA, 1 mM benzamidine, 0.01% Triton-X-100 and 0.1 mM phenylmethanesulfonyl fluoride, and assayed for superoxide. 12 gel filtration column (Pharmacia) equilibrated in the above buffer. Fractions containing p21 ^{WAF1 were detected} by Western blot using the p21 ^WAF1- specific monoclonal antibody Ab-1 (Oncogene Sciences), concentrated to 200 μg / ml and stored at -70 ° C.

GST-Rb - An E. coli expression construct, the the hyperphosphorylation domain of pRb (amino acids 773-924) was placed on a glutathione Sepharose column according to the manufacturer's instructions (Pharmacia).

Peptide precipitation of Cdk4 and cyclin D1

A 20 amino acid peptide ^library covering the entire sequence of p21 ^WAF1 ( 1 ) was screened for Cdk4 / cyclin D1 interaction peptides. Peptide (1.5 μg) was diluted in 100 μl PBS and incubated with 10 μl packed streptavidin agarose beads (Sigma) for 1 hour at room temperature. Unbound peptide was removed by extensive washing with PBS and the beads plus bound peptide were incubated for 1 hr at 4 ° C with reticulocyte lysate containing either Cdk4 or cyclin D1 labeled with [ ³⁵ S] -methionine. The beads were washed three times with 1.25 x PBS containing 0.2% Triton-X-100 and in the presence of 0.125 M Tris-HCl, pH 6.8 containing: 4% (w / v) SDS , 20% by volume glycerol and 200 mM DTT, cooked. The bound protein was analyzed by SDS-PAGE, followed by autoradiography and quantification of the ³⁵ S-labeled protein using a bio-imager and whole-band analysis software (Millipore).

enzyme tests

Phosphorylation of GST-Rb-Cdk4 activity was measured using the cyclin D1-Cdk4-containing insect cell lysate described above. Extract (1 μl) was added to a final reaction volume of 10 μl containing: 50 mM Hepes, pH 7.4, 10 mM MgCl 2, 2.5 mM EGTA, 1 mM DTT, 10 mM β-glycerophosphate, 1 mM NaF, 10 mM PKI, 50 μM ATP containing [ ³² P] -ATP (1,000 cpm / pmol) and 0.5 μg (GST-Rb) added. The experiments were started by the addition of the GST-Rb substrate, incubated at 30 ° C for 10 minutes (the incorporation of ³² P into GST-Rb was linear for 15-20 minutes) and by adding SDS-PAGE sample buffer and 4 minutes heating to 95 ° C completed. The samples were analyzed by SDS-PAGE on 12% gels analyzed, followed by autoradiography and quantification using a bio-imager.

Peptide phosphorylation

The biotinylated peptides (1 μg) were incubated for 30 minutes at 30 ° C in a final volume of 20 μl containing: 50 mM Hepes, pH 7.4, 10 mM MgCl ₂ , 2.5 mM EGTA, 1 mM DTT, 10 mM β-glycerophosphate, 1 mM NaF, 10 mM PKI, 50 μM ATP containing [ ³² P] -ATP (6,000 cpm / pmol) and either 1 μl cyclin D1-Cdk4 insect cell lysate, 1 μl uninfected insect cell lysate or 0.02 mU protein kinase C plus 0.5 mM CaCl ₂ , 100 mg / ml phosphatidylserine and 20 mg / ml diacylglycerol. The reactions were stopped by heating at 60 ° C for 5 minutes, and streptavidin-agarose beads were added (10 μl packed cell volume, washed with 3 × PBS) and incubated by shaking at 40 ° C for 30 minutes. The beads were washed extensively with PBS containing 3% by volume of Tween-20 and the incorporation of radioactivity into the peptides was determined by Cerenkov counting.

Cell cycle measurements

Carrier-bound peptides were created for delivery into proliferating HaCaT cells (see 9 ). The cells were seeded on 30 mm culture plates and grown to 50% confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% by volume fetal calf serum (FCS). Peptides were added to the medium and the cells were incubated for 24 hours. During the last 30 minutes of incubation, cells were pulsed in the presence of 15 μM BrdU. The cells were trypsinized, fixed in absolute alcohol and prepared for FACS analysis using a single-laser flow cytometer (Becton-Dickinson, FACScan) as previously described.

pRb phosphorylation in HaCaT cells

HaCaT cells were grown on 30 mm culture plates at 25% confluence in DMEM with 10% FCS inoculated. The FCS was withdrawn after 24 hours, and the cells were starved for 72 hours. At the end of this period was the medium with 10% FCS and carrier-bound Peptides supplemented. Samples were over taken a period of 24 hours, and the cells were in RIPA buffer (50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 1.0% by volume NP-40, 0.5% (W / v) DOC, 0.1% (w / v) SDS, 1mM PMSF, 0.1mg / ml aprotinin and 0.5 mg / ml leupeptin) for 30 minutes at 4 ° C. The phosphorylation stages of pRb were, as previously described, by Western blot analysis determined (Fahraeus et al. (1996)), with the exception that the blot with a polyclonal pRb antibody was probed (C-15, Santa Cruz).

results

Peptide binding assay for cyclin D1 and Cdk4

Using a series of synthetic peptides containing the entire sequence of p21 ^WAF1 ( 1 ), the inventors investigated whether these peptides could mimic the full-length p21 ^WAF1 protein by forming a stable complex with either cyclin D1 or Cdk4. If peptide-binding mimetics of the p21 ^WAF1 protein could be identified, this would help identify the minimal binding motif of the p21 ^WAF1 protein required for cyclin D1-Cdk4 holoenzyme inhibition and to find out whether p21 ^WAF1 was present targeted the cyclin or kinase subunit. This would also define a system to use small peptides to study the p21 ^WAF1 protein ^response mechanisms and to create mimicking drugs.

The peptide binding assay involved quantifying the amount of ³⁵ S-labeled cyclin D1 or Cdk4 that bound specifically to biotinylated peptides trapped on streptavidin-coated agarose beads. The peptide-coated beads were added to extracts containing either ³⁵ S-labeled cyclin D1 or Cdk4 which was translated in vitro, the beads were washed extensively to remove unbound proteins, and the bound cyclin D1 or Cdk4 was analyzed by SDS -PAGE, quantified by autoradiography and bioimaging. This is referred to below as the peptide precipitation test and has previously been used to demonstrate evolutionary conservation of p21 ^WAF1 binding to PCNA (Ball & Lane (1996)).

A small peptide derived from amino acids 46-65 in the N-terminal domain of p21 ^WAF1 binds directly to Cdk4

Using the peptide precipitation assay, peptide 4 (from the N-terminal domain of p21 ^WAF1 ) ^bound specifically to Cdk4, but not to cyclin D1 ( 1 ). This interaction is physiologically important since it has previously been suggested that the CDK interaction domain of the p21 ^WAF1 protein would localize to the N-terminal domain of the molecule (Chen et al., (1995); Harper et al., (1995); Luo et al al., (1995)). In particular, deletions (Nakanishi et al. (1995a)) or mutations (Goubin and Ducommun (1995)) in the region of amino acids 45-71 compromise the ability of full-length p21WAF1 to interact with Cdk2. Whether this loss of p21WAF1 binding function is due to (i) Mutation / deletion of residues directly involved in CDK binding or whether (ii) mutations / deletions caused conformational changes in p21 ^WAF1 that prevent stable binding to CDK has not yet been demonstrated. Here, the inventors unequivocally show that residues 46-65 are directly involved in the binding of p21 ^WAF1 to Cdk4 and that they alone are capable of forming a stable complex with Cdk4 in the absence of cyclin D1. This in turn is direct evidence that the N-terminus of p21 ^{WAF1 contains} a kinase binding domain.

A small peptide derived from amino acids 16-35 in the N-terminus of p21 ^WAF1 binds directly to cyclin D1

The inventors could also define a second and distinct N-terminal interaction site on the p21 ^WAF1 protein; in this case, a region of p21 ^WAF1 capable of binding to cyclin D1 but not to Cdk4 ( 1 ). Peptide 2 comprises amino acids 16-35 of p21 ^WAF1 and is in the minimum region required for in vivo DNA synthesis inhibition, which lies between residues 17-71 (Nakanishi et al., 1995a). The results of the inventors could explain an obvious contradiction to which Nakanishi et al. (1995a) who found that N-terminal mutations in the p21 ^WAF1 protein that are outside the CDK interaction domain, although insufficient to prevent binding to the kinase, were sufficient to ^attach p21 ^WAF1 to it to prevent acting as a growth suppressor when transfected into proliferating cells. In particular, direct peptide binding data ( 1 ) suggesting that the N-terminal motif in the p21 ^WAF1 protein mediates cyclin D1 binding would be an essential step in the mechanism by which the p21 ^WAF1 protein acts as a growth ^suppressor .

A novel cyclin D1-Cdk4 binding motif is located at the C-terminus of the p21 ^WAF1 protein

The specificity of the peptide precipitation assay in defining the domain of p21 ^WAF1 protein required to bind either cyclin D1 or Cdk4 ( 1 ), showed that the use of peptides to study potential interactions between p21 ^WAF1 and cyclin D1 complexes would be very informative. However, the inventors were fascinated that peptides from the C-terminus of the p21 ^WAF1 protein (peptides 10 and 11) could form stable complexes with both Cdk4 and cyclin D1 ( 1 ), since peptide 10 was treated with the method described by Warbrick et al. (1995) is equivalent to p21PBP peptide and represents the region of p21 ^WAF1 that binds to the replication / repair protein PCNA. The inventors can not rule out the possibility that endogenous cyclin or CDK present in the reticulocyte lysate might bind to the labeled human protein by forming a bridge to the peptide. However, since peptide 2 and peptide 4 precipitate either cyclin D1 or Cdk4, this seems unlikely.

These results suggest that the p21 ^WAF1 protein could interact with both PCNA and cyclin-CDK complexes through the same binding motif. However, peptide 11 binds to both Cdk4 and cyclin D1, but not to PCNA ( 1 (Warbrick et al., 1995; Ball & Lane, 1996); whereby the PCNA binding site is decoupled from the cyclin-CDK binding motif in the C-terminus of p21 ^WAF1 .

Considering that the inventors identified three distinct motifs of the p21 ^WAF1 protein that specifically bind to cyclin D1 and / or Cdk4, the inventors now investigated whether they ^mimicked the p21 ^WAF1 protein by inhibiting kinase activity.

The cyclin D1 binding ^peptide from the N-terminal domain of p21 ^WAF1 and the cyclin / CDK binding ^peptide from the C-terminus of p21 ^WAF1 inhibit the activity of Cdk4

To determine whether any of the p21 ^WAF1 peptides has Cdk4 inhibitory activity, the inventors independently tested their ability to prevent pRb phosphorylation during cyclin D1-Cdk4 assays in vitro ( 2 ). Peptides 2, 8, 10 and 11 inhibited cyclin D1-Cdk4 activity when added to the 17 μM assay, whereas buffer alone and the remaining peptides had no dramatic effect on Cdk4 activity. The cyclin D1 binding peptide (peptide 2) inhibited kinase activity by about 80%, and peptides 10 and 11, which bound both Cdk4 and cyclin D1, completely inhibited enzyme activity at this concentration. Thus, there is a correlation between the ability of the peptides to bind to Cdk4 and / or cyclin D1 and to inhibit Cdk4 kinase activity.

However, this correlation does not persist in the case of kinase binding peptide 4. This peptide maps to the CDK interaction site ( 8th ; Goubin & Ducommun (1995); Nakanishi et al. (1995a)), and there was speculation that a peptide from this domain capable of interacting with CDK would mimic full-length p21 ^{WAF1 inhibitory activity} , thus providing a model for the design of new molecules that would advance the cell cycle Inhibition of G1-cyclin CDKs could stall. Despite the high affinity for Cdk4, peptide 4 showed no inhibitory activity when compared to cyclin D1-Cdk4 assays in concentrate was added up to 35 μM. ^Thus, the inventors' data of both p21 ^WAF1 peptide ^binding data and inhibitory properties indicate two new small domains of p21 ^WAF1 protein as potential candidates for low molecular weight mimetics; an N-terminal motif from amino acids 16-35 (peptide 2) and a C-terminal motif from amino acids 141-160 (peptide 10).

It is therefore possible that peptide 4 block p21 binding and its activity as an inhibitor could prevent. Therefore, cells treated with peptide 4 may be expected they would even in the presence of competing signals, normally Communicating cell cycle arrest or apoptosis, proliferating. peptide 4 can therefore be used to access cells by adding the peptide to reversibly immortalize to the cells. That represents another Tool for exploring cellular Mechanisms to control the cell cycle are ready and able as well useful be to fight cell loss in diseases associated with the loss be associated by cells, such as. AIDS or degenerative diseases, including MS, dementia or degenerative muscle diseases, such as z. Muscle wasting (MD), including Duchenne MD.

The C-terminal p21 ^WAF1 peptide is a stronger inhibitor of Cdk4 kinase activity than the N-terminal cyclin D1 binding peptide

The inventors carried out more detailed studies to determine I _0.5 for peptides 2, 8 and 10, using peptide 4 as a negative control ( 3 ). The inventors found that peptide 10 (and peptide 11, data not shown) was a potent inhibitor of Cdk4 activity with an I _0.5 of 0.1 μM, peptide 2 was also a good inhibitor with an I _0.5 of 2 μM. Peptide 8 provided only little inhibition and relatively high peptide concentrations were necessary to achieve 50% inhibition. These data support the ability to use peptide 2 or peptide 10 to mimic the CDK ^{inhibitory activity of} the full-length p21 ^WAF1 protein.

p21 ^WAF1 protein and inhibitory peptides compete for the same binding site on Cdk4 kinase

To determine whether the Cdk4 inhibitory peptides, 2 and 10, acted on sites on Cdk4 and cyclin D1 also used by p21 ^WAF1 , the inventors performed peptide precipitation assays in the presence and absence of purified full length his-p21 ^WAF1 to find out if it competed with the peptides for binding.

The ability of p21 ^{WAF1 to disrupt} peptide 2 (A) and peptide 10 binding (B & C) to Cdk4 and / or cyclin D1 was determined by performing the peptide precipitation assay from reticulocyte lysates in the presence of 0, 0.5, 2 μg p21 ^WAF1 determined.

The data suggest that binding of the p21 ^WAF1 protein to cyclin D1 and Cdk4 prevents binding of both peptide 2 and peptide 10. These data allow for two interpretations, (i) the peptides could compete for binding at the same site as p21 ^WAF1 , or (ii) binding of either p21 ^WAF1 or peptide could cause a conformational change in the cyclin or CDK and prevent further binding. From these experiments, it is not clear whether peptides 2 and 10 act at the same site (s). The difference in peptide precipitation data indicates that at least one of the sites is unique, since peptide 10 can precipitate both Cdk4 and cyclin D1, whereas peptide 2 can precipitate only cyclin D1.

Data to support the hypothesis that peptide 10 and p21 ^WAF1 protein compete for the same binding site during kinase inhibition are based on the use of a peptide-10 mutant (containing a point mutation resulting in a change of RA in the remainder of the 15 Peptide 10, which is equivalent to residue 155 of the full length protein), which loses> 60% of its inhibitory activity (see below) but retains its binding function.

To determine whether inhibition of Cdk4 by p21 ^WAF1 still had partial binding ^activity by addition of a peptide-10 mutant, the R-to-A mutant (residue 15 of peptide 10), which was no longer an efficient inhibitor , can be decreased, increasing peptide concentrations (1, 5, 17 & 34 μM) in the presence of a fixed concentration of p21 ^WAF1 (50 μM) were added to the cyclin D1-Cdk4 GST-Rb phosphorylation assay.

The experiment showed that increasing concentrations of mutant peptide-10 were able to block the inhibitory activity of full-length p21 ^WAF1 , suggesting that peptide 10 binds to the site (s), which ^inhibits the subsequent binding of p21 ^WAF1 blocks and therefore functions by a mechanism similar to that of the full length protein.

The inhibitory peptides are not Cyclin D1-Cdk4 substrates

In contrast to the p107 peptide, which appears to inhibit the ability of Cdk4 to phosphorylate pRb by acting as an alternative substrate (Zhu et al. (1995)), p21 ^{WAF1 was} not reported to be a substrate for the cyclin Act D1-Cdk4 complexes (and the inventors be confirm these observations, 4 ). However, it is possible that by using p21 ^WAF1- based proteins instead of full-length proteins, the inventors have inadvertently generated phosphorylation sites that would not normally be exposed on the surface of the protein. The peptides could therefore act as competing substrates as opposed to inhibitors of catalytic activity. Both Peptide 2 and Peptide 10 contain a number of potential phosphorylation sites, and the inventors were able to demonstrate that Peptide 10 is a potential substrate for a range of protein kinases (data not shown), including Protein Kinase C (PKC), which was used as a control kinase ( 4 ). In fact, neither peptide 2 nor peptide 10 were substrates for cyclin D1-Cdk4 under conditions in which 2.4 nmoles of ³² P per nmol of GST-Rb were incorporated. However, under the same conditions, peptide 10 was a very good substrate for PKC with 0.82 nmol of ³² P incorporated per nmol of peptide ( 4 ). There was a low level of incorporation into peptide 2, but since this was also present in assays using lysate from uninfected insect cells, this must be due to the small size of endogenous protein kinase (s). It therefore appears that the peptide inhibitors are not competing substrates but act as if to block the catalytic activity in a mechanism similar to that of p21 ^WAF1 .

The peptides are not efficient inhibitors cyclin B-Cdc2 kinase activity

Harper et al. (1995) showed that p21 ^{WAF1 is} not a universal CDK inhibitor, but that it has selectivity for the G1 and S phase cyclin CDK complexes. When they compared the ability of p21 ^{WAF1 to inhibit} cyclin B-Cdc2 acting at the G2 / M junction and cyclin D2-Cdk4 acting during G1, they found that the I was _0.5 for Inhibition of cyclin B-Cdc2> 600 fold higher than I _0.5 for inhibition of cyclin D2-Cdk4 using purified recombinant proteins. The inventors considered the effect of adding their two cyclin D1 Cdk4 inhibitor peptides to cyclin B-Cdc2 and Cdk2 cyclin E assays at concentrations of up to 20 μM and found that neither peptide 2 nor peptide 10 had a significant effect on Cdc2-cyclin B histone H1 kinase activity. Cdk2-cyclin E was however inhibited by peptide 10, indicating that peptide can inhibit 10 other G ₁ cyclin-Cdk complexes. Therefore, the p21 ^WAF1- based peptide inhibitors appear to have a specificity equivalent to that of the full-length protein.

Around to determine if peptides 2 and 10 are cyclin B-Cdc2 and cyclin E-Cdk2 kinase activity assays were performed using Sf9 cell lysates carried out, that co-expressed human cyclin B and Cdc2. The conditions were identical to those used in the experimental procedures for cyclin D1-Cdk4 with the exception that histone H1 (0.5 μg / test) as Substrate for Cyclin B-Cdc2 was used. Cyclin D1-Cdk4, cyclin B-Cdc2 and cyclin E-Cdk2 were in the presence of increasing concentrations of peptide 2 (0.25, 3, 10 and 40 μM) and Peptide 10 (0.1, 0.5, 5, 20 μM) tested.

The kinase inhibition motif of peptide 10 differs from the PCNA binding site

The inventors have shown that peptide 10 is a particularly potent inhibitor of cyclin D1-Cdk4 activity, with an I _0.5 of 0.1 mM, which is 20-fold more potent than peptide 2, a peptide derived from the region of p21 ^WAF1 previously associated with growth ^arrest (Chen et al., 1995; Nakanishi et al., 1995a). The inventors have also shown that a peptide (peptide 4) comprising the CDK interaction site of p21 ^WAF1 (Goubin & Ducommun (1995); Nakanishi et al. (1995a)), although capable of binding to Cdk4 and to form a stable complex, has no detectable activity as a cyclin D1-Cdk4 inhibitor. Peptide 10 therefore appears to be the best candidate for the development of a small peptide mimetic with high potency. Peptide 10 has previously been shown to form a specific high affinity and reversible interaction with PCNA (Ball & Lane (1996)), and this peptide is sufficient to partially inhibit the function of PCNA during SV40 replication 50% inhibition at a concentration of about 7 mM (Warbrick et al., 1995). The PCNA interaction ^domain of p21 ^WAF1 was mapped, and it was found that the important residues were amino acids 144-151 (QTSMTDFY, Warbrick et al (1995), Ball & Lane (1996)). Although the extreme C-terminal peptide (peptide 11) has amino acid residues that are important for binding to and inhibiting Cdk4 (see 1 and 2 ), it can not bind PCNA (Warbrick et al. (1995); Ball & Lane (1996)). These results indicate that the kinase inhibition and PCNA binding ^{motifs are} different in the C-terminus of p21 ^WAF1 , but it does not exclude the possibility that an interaction between p21 ^WAF1 and PCNA or cyclin / kinase may require some common amino acids. Therefore, it is important to identify the precise inhibitory motif in the C-terminus of p21 ^WAF1 and to find out whether it overlaps with or differs from the PCNA interaction ^domain . To investigate this question, the inventors have chosen two approaches; they synthesized (i) a series of peptides that stretched 4 amino acids in each direction along peptide 10 (size scan; 7 ), and (ii) one A set of peptides based on peptide 10 where each residue was sequentially mutated to alanine (alanine scan; 6 ). The ability of the peptides to inhibit Cdk4 activity in vitro in each of the two series was then determined. Using the size scan, the inventors found that peptide inhibitory activity required amino acids 156-160, while amino acids 148-155 were dispensable. This decouples the kinase inhibition motif from the PCNA binding motif.

With the alanine scan, the inventors defined the critical residues for inhibition by demonstrating that a stretch of only 5 amino acids was essential for activity, with a single conservative point mutation on one of two hydrophobic residues, completely eliminating the inhibitory activity of peptide 10 ( 6 ). The essential amino acids are RRLIF (amino acids 155-160), where the bold letters are essential to the activity and the underlined residue significantly contributes to the inhibitory activity.

During testing in the peptide precipitation test, mutation of the first R of this motif to A (aa 155 of full length p21 ^WAF1 ) partially retained its ability to bind both Cdk4 and cyclin D1, while mutations from L or F to A retained the affinity significantly reduced Cdk4 and cyclin D1, and mutations of the second R or I did not show any effect on binding (data not shown). Therefore, the RA mutant was used in competition tests. The fact that a single point mutation on one of the two hydrophobic residues (the L or F residue) completely eliminated the inhibitory activity indicated that the inhibition was due to a specific interaction at hydrophobic key residues. The mapping data also explain why both peptide 10 and peptide 11 are good inhibitors of cyclin D1-Cdk4 activity ( 2 ), since both contain the inhibitory motif. It therefore appears that the inhibitory part of peptide 10 would not overlap with the PCNA binding site because they have no common amino acid residues.

A single amino acid substitution in peptide 10 makes it a stronger inhibitor, thus approximating the specific activity of a full-length p21 ^WAF1 protein

While conducting the alanine canine experiments, the inventors noted that one of the mutant peptides (DA at position 9 of peptide 10 or 149 of the full-length protein; 6 ) the peptide appeared to be a better inhibitor of cyclin D1-Cdk4 activity. The inventors determined I _0.5 for this peptide and compared it to peptide 10, purified full-length his-p21 ^WAF1, and a peptide derived from the tumor suppressor protein p16 INK4, which was recently reported to be the cyclin D1 receptor. Inhibit Cdk4 activity in vitro and prevent the progression of the cell cycle (Fahraeus et al., (1996)). The DA mutation reduces I _0.5 from 100 nM to 46 nM ( 7 ). Now, when compared to the p16INK4-based peptide that has an I _0.5 of 16.3 μM 7 ), the inventors have now produced a peptide that is about 350 times more active than a Cdk4-inhibiting compound. In fact, the inventors now begin to ^{approximate the potency} of p21 ^WAF1 itself, which has an I _0.5 of 11 nM in the insect ^{cell lysate assay} ( 7 ). This value is in the same range as the 40 nM Ki for p21 ^WAF1 , which was reported by Harper et al. (1995) for the inhibition of cyclin D1-Cdk4 in Sf9 cell lysates. Compared to the full-length protein, the mutant peptide 10 only has a 3.5-fold lower specific activity than a kinase inhibitor in crude lysates. Why a mutation DA at this position, which is located a good deal outside the domain, which is shown to be essential for the activity, leads to a reduction of I _0.5 , is not known. It seems likely that it involves the presentation of the inhibitory motif rather than a direct role for this residue in the inhibition, as it does not appear that this mutation would increase the affinity of the peptide for either Cdk4 or cyclin D1 (data not shown). ,

The results indicate that peptide 10 could be used as a model to base small peptide mimetics of p21 ^WAF1 on it, and the inventors have provided evidence that changes in the peptide structure or presentation of the active residues can lead to the formation of a peptide inhibitor, which resembles the full-length p21 ^{WAF1's strength} as a cyclin D-Cdk4 inhibitor.

Results for the C-terminal peptide (peptide 10)

An eight amino acid peptide is sufficient to the cyclin D-Cdk4 activity to inhibit

Having identified residues that were crucial for the inhibition of cyclin D1-Cdk4 by peptide 10, the inventors determined whether these residues were sufficient for inhibition or whether in the context of a larger peptide. had to be presented. Surprisingly, the eight-amino acid peptide, KRRLIFSK, retained the ability to completely inhibit cyclin D1-Cdk4 activity and prevent phosphorylation of pRb ( 6 ). However, the I _0.5 for the truncated peptide was about 1000-fold higher than that of the full-length peptide (I _0.5 for the truncated peptide was about 100 μM). This was not an unexpected result, as other studies have shown a loss of performance after reducing the length of the bioactive peptides. However, it might be possible the inhibitori to improve the activity of the peptide by manipulation of the non-essential residues as described by Lin et al. (1995), in an elegant series of experiments aimed at reducing the polypeptide formed in the anterior chambers of the heart.

Peptide 10 works in cell systems

The introduction of p21 ^WAF1 cDNA into human brain, lung and colon cancer cell lines leads to cell growth suppression (El-Deiry et al., 1993). In addition, during a radiation-induced G ₁ arrest in human fibroblasts, p21 ^WAF1 protein ^{levels increase} in a p53-dependent manner, resulting in a strong inhibition of G ₁ -cyclin CDKs as well as failure of cells to enter S phase , (Dulic et al. (1994); Harper et al. (1995)). In order for peptide 10 to function as a realistic template for the design of novel antiproliferative drugs, it must be capable of ^mimicking the CKI activity of p21 ^WAF1 as a growth ^suppressor in a cellular background. The inventors, as well as others, have recently shown that a 16 amino acid sequence can act from the homodomain of the antennapedia protein as a carrier for peptides with biological activity, translocating them across the plasma membrane and allowing them to interact with their target molecules ( Fahraeus et al (1996); Hall et al. (1996)). To determine if peptide 10 retained its biological activity when introduced into tissue culture cells, the inventors synthesized it directly on the carrier peptide and added it to a culture of proliferating asynchronous human HaCaT cells derived from keratinocytes. The bound peptide (called Peptide-I; 9 ) contained a mutation of M to A at position 7, resulting in the elimination of its activity as a PCNA-binding peptide (Warbrick et al., 1995; Ball & Lane (1996)), and allowed the inventors to have PCNA-independent effects of the peptide to study the normal cell cycle.

Peptide-I was added to the culture medium at a concentration of 25 μM, the cells were fixed 24 hours later and then analyzed by fluorescence-activated cell sorting (FACS). G ₁ , S and G ₂ phase distribution of untreated and peptide I-treated cells was tested using bromodeoxyuracil (BrdU). The number of cells entering the S phase in the presence of peptide I was dramatically reduced and the G ₁ population showed a concomitant increase. This suggests that peptide- ₁ mimics the ability of full-length p21 ^WAF1 to act as a growth ^suppressor by inducing G1 cell cycle arrest.

To ascertain whether Peptide-I acted as a growth ^inhibitor by preventing the phosphorylation of pRb in a manner analogous to that of p21 ^WAF1 , the inventors used serum starvation to produce a synchronous population of HaCaT cells. Peptide-I was added to the cells at the same time as they were released from serum starvation, and samples from treated and untreated cells were taken over a period of 24 hours. The phosphorylation status of pRb was monitored by a gel retention test. When serum was added to the starved cells, pRb was hyperphosphorylated for between 12 and 15 hours, but in the presence of peptide I, pRb remained hypophosphorylated. Therefore, Peptide-I leads to a G ₁ arrest in human HaCaT cells by preventing the phosphorylation of pRb.

The inventors used an identical experimental approach to introduce (i) the bioactive truncated peptide 10 and (ii) a control peptide 10 which lacked essential residues for CDK inhibition in HaCaT cells. The inventors found that peptide II effectively promoted G ₁ phase arrest and completely prevented the phosphorylation of pRb when added at 50 μM ( 9b ). However, peptide III lacking the last 4 amino acids of peptide 10 (LIFS) had no detectable effect on the ability of HaCaT cells to enter S phase. It is interesting that truncated peptide 10, when coupled to a carrier peptide (peptide II) and introduced into cells, is only 2 times less active than growth suppressor than peptide I (see above for in vitro data ). Binding of the truncated peptide 10 to the carrier peptide may promote a more suitable inhibitory conformation than the I _0.5 for carrier-bound truncated peptide 10 (peptide II is approximately 50-fold less in vitro than that of the free peptide 10 (data not shown)). ,

peptide 10 was added to Rb-negative cells and the results support its imitation the full-length protein, d. H. it can mimic its biological activity as a cell cycle inhibitor. Peptide 10 was found to be cell cycle arrest in pRb-negative and in pRb-positive cells causes.

Using Soas2 cells, the introduction of peptide 1 (peptide 10 bound to penetratin and mutated to prevent PCNA binding) results in an increase in the population of cells in the G ₁ phase.

discussion

Synthetic peptides or peptide mimetics have proven useful in studying the biochemical regulation of enzymes and proteins. they also provide models for designing novel antiproliferative agents that target the amplified enzymatic pathways or proteins that are activated in human tumors (Powis (1992), Gibbs & Oliff (1994)). Peptides which have also been shown to be efficacious for components of the cell cycle mechanism include: FTIs that inhibit farnesyl protein transferase to prevent activation of Ras (Gibbs et al., 1994); Ras effector domain peptides that can inhibit its biological function (Moodie & Wolfman (1994); Rodriguez-Viciana et al. (1994)); Polypeptides carrying SH2 / SH3 domains which should theoretically inhibit the growth of tumors with activated tyrosine kinases (Pawson & Schlessinger (1993), Yu et al., (1994)) and p16INK4-derived peptides containing the Inhibit cyclin D-CDK complex activity and thereby activate pRb-dependent cell cycle arrest (Fahraeus et al., (1996)).

The Inactivation of the tumor suppressor protein p53 is a common event in the development of human neoplasia (Hollstein et al., 1991). The p53 protein is a key player in an inducible cell cycle checkpoint pathway, the in response to DNA damage and nucleotide pool disorders activated (Lane (1992); Agarwal et al. (1995)). The reactivation this metabolic pathway could therefore a way to discover new antiproliferative drugs provide. A number of mechanisms may be used for functional inactivation of the p53 pathway to lead, including the inactivation of downstream effector molecules of p53, such as The cyclin kinase inhibitor p21WAF1 (Deng et al. (1995); Waldman et al. (1995)). Recent developments have shown that reactivation of the p53 pathway in some human Tumors by activating the biochemical function of the endogenous mutant p53 protein possible may be (Halazonetis & Kandil (1993); Hupp et al. (1993)), possibly under Use of small peptides as lead compounds for drug design (Hupp et al., 1995)) or by reintroduction of the wild-type p53 gene by adenovirus vectors (Eastham et al., 1995). In general However, the pharmacological restoration of biochemical Function of a protein that passes through its normal activity Mutation of its amino acid sequence lost, more difficult than the inhibition of a biochemical Function (Gibbs & Oliff (1994)). Therefore, it can prove to be more productive, alternative approaches to restore the activity of the p53 pathway to choose, such as B. mimicking the inhibitory activity of the downstream effector molecule p21WAF1, the one growth arrest alone primarily by its interaction with the G1-cyclin CDKs (El-Deiry et al. (1993); Eastham et al. (1995); Harper et al. (1995)).

The determination of the minimal domain of p21 ^WAF1 , which can inhibit CDK function, and finding out if such a domain can function in high efficiency isolation are two important goals to be achieved in order to determine whether p21 ^WAF1 is a realistic template for use in antiproliferative drug design research. Prior to the inventors' studies, the minimal sequence of p21 ^WAF1 , which had inhibited CDK function in vitro, was the N-terminal domain (residues 1-75) (Luo et al., 1995). While peptides derived from this N-terminal domain were recently shown to antagonize the ability of p21 ^WAF1 to inhibit cyclin E-Cdk2 complex activity, suggesting that this domain interacts with the kinase (Chen et al., (1996)), no data has yet been presented on the direct interaction of small peptides with either cyclin or CDK. In addition, there was no evidence to suggest that a small peptide derived from p21 ^WAF1 would actually be biologically active as a CDK inhibitor. Since the cyclin D1-Cdk4 complexes and the related isoforms are essential for the passage of the G1 phase, the inventors have used a series of small synthetic peptides based on the sequence of p21 ^WAF1 to (i) determine whether there are Cdk4 inhibitory peptide mimetics and whether these are of great effectiveness, and (ii) to probe the mechanism by which the p21 ^WAF1 protein inhibits cyclin D1-Cdk4 activity.

A model for the inhibition of cyclin D1-Cdk4 by p21 ^WAF1

Two different peptides from the N-terminal domain of p21 ^WAF1 interacted with either Cdk4 or cyclin D1 to form stable complexes. One peptide bound to Cdk4, but did not inhibit its activity while the second bound specifically to cyclin D1 and had a potent inhibitory effect on cyclin D1-Cdk4 activity. Cdk4 binding peptide 4 (residues 46-65) corresponded to a putative Cdk2 binding domain of p21 ^WAF1 previously defined using p21 ^WAF1 deletion constructs (Nakanishi et al. (1995a) and alanine mutation analysis (Goubin & Gucommun (1995) The inventors have found that this region of p21 ^{WAF1 is} indeed directly involved in CDK binding but has no Cdk4 inhibitory activity ( 2 and 3 ). These data explain why certain N-terminally deleted p21 ^WAF1 constructs that still contain the CDK binding site are unable to effectively inhibit cell growth (Nakanishi et al., 1995a).

The second N-terminal peptide that bound to cyclin D1 inhibited cyclin D1-Cdk4 activity strong by a new mechanism (see below). The mechanism for p21 ^WAF1 inhibition of cyclin-CDK complexes has been poorly understood since it was not clear whether the p21 ^WAF1 protein inhibited by cyclin and / or kinase subunit binding. Cdk2 binds very weakly to p21 ^WAF1 in the absence of cyclin, and the affinity of G1-CDKs for p21 ^WAF1 increased markedly when CDK was associated with a cyclin (Harper et al., 1995), suggesting that cycline may be a play important role in the p21 ^WAF1 inhibition of CDK activity. Whether, however, a CKI, such. For example, p21 ^WAF1 and p27KIP1 can interact directly with cyclin is controversial (Toyoshima & Hunter (1994); Harper et al. (1995)). However, a recent study suggested that p21 ^WAF1 may interact directly with a number of cyclins in the absence of CDK (Fotedar et al., (1996)). The inventors show here that a small peptide composed of residues 16-35 (peptide 2) forms a stable complex with cyclin D1 and that this peptide alone is a potent inhibitor of Cdk4 activity, with an I _{0 , 5} of 2 mM. This peptide falls into the growth suppression region (residues 17-71) described by Nakanishi et al. (1995a). This is the first time that a putative cyclin binding site on p21 ^{WAF1 has been} identified and that a small synthetic peptide representing this domain has been shown to be sufficient to mimic the full length p21 ^WAF1 protein as a CDK inhibitor.

The Fact that the cyclin D1-Cdk4 activity is due solely to interaction with the cyclin subunit can be inhibited, suggests suggest that either (i) conformational changes in cyclin D1 lead to Inhibition of catalytic Cdk4 activity may lead to (ii) peptide 2's interaction interferes with cyclin D1 with Cdk4 or that (iii) peptide 2 interacts with cyclin D1-Cdk4 interferes with its substrate pRb.

The outlook for the design of ^{small molecule} mimetics of p21 ^WAF1 is more viable given that the cyclin D1 binding ^peptide alone can inhibit kinase function, suggesting that the prior presence of a p21 ^WAF1 protein ^binding to the kinase Subunit binds, for the inhibition of the kinase function is not necessary. In addition, the amino acid residues ^conserved between p21 ^WAF1 and its close relative p27KIP1 (Polyak et al., 1994; Toyoshima & Hunter, 1994) are clustered in the N-terminal domain, with the regions corresponding to peptides 2 (FIG. 65% identical) and peptide 4 (50% identical), containing the majority of the conserved amino acids. This suggests that inhibition of Cdk4 activity by interaction with the cyclin D subunit may be a common mechanism used by both p21 ^WAF1 and p27KIP1.

A new C-terminal p21 ^WAF1- cyclin D1-Cdk4 inhibition domain

During the inventors' studies, they also found that a peptide (peptide 10) from the C-terminal domain of p21 ^{WAF1 was} a potent inhibitor of cyclin D1-Cdk4 activity in vitro. The inhibitory motif was identified and distinguished from the PCNA interaction site, which is also located in the C-terminal domain of p21 ^WAF1 (Chen et al., 1995; Luo et al., 1995; Warbrick et al., 1995 ) Ball & Lane (1996)). The results of the inventors are in contrast to previous studies which found that cyclin Cdk2 inhibitory activity is restricted to the N-terminal domain of p21 ^WAF1 alone when each half is expressed separately (Chen et al., 1995; Luo et al., (1995)). The reasons for this discrepancy may include the following: (i) the use of C-terminally-labeled p21 ^WAF1 in expression ^vectors to purify p21 ^WAF1 constructs (Luo et al. (1995)) showing the local structure at the C-terminus could have influenced P21 ^WAF1 ; (ii) transfection of constructs containing only the C-terminal half of p21 ^WAF1 (Chen et al., 1995; Luo et al., 1995), demonstrating folding into the correct native conformation, excluding the identification of the can make difficult the new inhibitory domain; (iii) by using peptides instead of the C-terminal constructs or the full-length p21 ^WAF1 protein, the inventors could have revealed sites that would not have been shown to the solvent in full-length native p21 ^WAF1 protein; (iv) it is possible that there may be slight differences in mechanism (s) used by p21 ^WAF1 to inhibit the cyclin-Cdk2 complexes and cyclin D1-Cdk4. Whether the C-terminal inhibitory motif defines a new physiologically relevant regulatory site on p21 ^WAF1 is currently being addressed. However, the potency of peptide 10 (I _0.5 = 0.1mM, only 10-fold lower than the full-length p21 ^WAF1 protein in these assays) and its ability to completely inhibit cyclin D1-Cdk4, indicates the high potency of peptide 10 ^Suggest that it is worth pursuing further studies of this region of the full-length p21 ^WAF1 protein.

Peptide 10 represents a potentially exciting drug design pathway, as it is by far the strongest peptide inhibitor of CDK activity discovered to date because it is> 150-fold better than the recently identified peptidomimetic of p16INK4 (Fahraeus et al., (1996)) and 20 times better than the N-terminal inhibiting p21 ^WAF1- derived peptide described by the inventors. The fact that the residues that are important for inhibitory activity are limited to a stretch of only five amino acids, indicates that contact in a single borderline region is sufficient to produce a very potent inhibitor of cyclin D1-Cdk4 activity, ^{thus becoming} a realistic template for the design of small molecules that mimic p21 ^WAF1 activity.

The fact that peptide 10 retains inhibitory activity when reduced to only eight amino acids (KRRLIFSK) enhances the incentive to use it as a template for rational drug design. In general, protein-protein interfaces are relatively large, based on the involvement of between 10-30 contact side chains on each interface, each contact region often being composed of residues scattered throughout the primary amino acid sequence (Davies et al., 1990). de Vos et al., (1992)). However, there is evidence that in some cases only a small subset of these side chains must be contacted for efficient binding (Kelly &O'Connel (1993), Cunningham & Wells (1994), Clackson & Wells (1995)). The discovery that a single eight amino acid peptide alone is sufficient to inhibit the activity of a key G ₁ -cyclin CDK, preventing pRb phosphorylation and producing a G ₁ cell cycle arrest in tissue culture cell systems, suggests that Interaction at only a small subset of the contact side chains is necessary for strong inhibition of cyclin D1-Cdk4 activity at the G1-S phase boundary. This makes cyclin D1-Cdk4 a realistic and exciting target for the design of small synthetic compounds that can act as antiproliferative agents.

bibliography

All References, no matter where in this document they are are incorporated herein by reference.

• Agarwal, et al. (1995). Proc. Natl. Acad. Sci. U.S.A., 92: 8493-8497.
• Baldin, et al. (1993). Genes Dev., 7: 812-821.
• Ball, K.L. and Lane, D.P. (1996). Biochem., 237: 854-861.
• Buckbinder, et al. (1995). Nature, 377: 646-649.
• Chen, et al. (1996). Oncogene, 12: 595-607.
• Chen, et al. (1995). Nature, 374: 386-388.
• Clackson, T. and Wells J.A. (1995). Science, 267: 383-386.
• Clarke, et al. (1993). Nature, 362: 849-852.
• Cunningham, B.C. and Wells, J.A. (1994). J. Mol. Biol., 234: 554-563.
• Davies, et al. (1990). Rev. Biochem., 59: 439-473.
• Deng, et al. (1995). Cell, 82: 675-684.
• de Vos, et al. (1992). Science, 255: 306-312.
• Dulic, et al. (1994). Cell, 76: 1013-1023.
• Eastham, et al. (1995). Cancer Res., 55: 5151-5155.
• El-Deiry, et al. (1993). Cell, 75: 817-825.
• Fahraeus, et al. (1996). Curr. Biol., 6: 84-91.
• Flores-Rozas, et al. (1994). Proc. Natl. Acad. Sci. USA., 91: 8655-8659.
• Gibbs, J.B. and Oliff, A. (1994). Cell, 79: 193-198.
• Gibbs, et al. (1994). Cell, 77: 175-178.
• Goubin, F. and Ducommun, B. (1995). Oncogene, 10: 2281-2287.
• Gu, et al. (1993). Nature, 371: 257-261.
• Halazonetis, T.D. and Kandil, A.N. (1993). EMBO J., 12: 5057-5064.
• Hall, et al. (1996). Curr. Biol., 6: 580-587.
• Harper, et al. (1993). Cell, 75: 805-816.
• Harper, et al. Tsai, L-H., Zhang, P., Dobrowolski, C.B., Connell-Crowley, et al. (1995). Cell, 6: 387-400.
• Hollstein, et al. (1991). Science, 253: 49-53.
• Hupp, et al. (1993). Nucl. Acids Res., 21: 3167-3174.
• Hupp, et al. (1995). Cell, 83: 337-345.
• Kastan, et al. (1991). Cancer Res., 51: 6304-6311.
• Kastan, et al. (1992). Cell, 71: 587-597.
• Kearsey, et al. (1995). Science, 270: 1004-1005.
• Kelley, R.F. and O'Connell, M.P. (1993). Biochemistry, 32: 6828-6835.
• Lane, D.P. (1992). Nature, 358: 15-16.
• Lowe, et al. (1993). Nature, 362: 847-849.
• Luo, et al. (1995). Nature, 375: 159-161.
• Lu, X. and Lane, D.P. (1993). Cell, 75: 765-778.
• Merritt, et al. (1994). Cancer Res., 54: 614-617.
• Miyashita, T. and Reed, J.C. (1995). Cell, 80: 293-299.
• Momand, et al. (1992). Cell, 69: 1237-1245.
• Moodie, S.A. and Wolfman, A. (1994). Trends Genet., 10: 44-48.
• Nakanishi, et al. (1995a). EMBO J., 14: 555-563.
• Nakanishi, et al. (1995b). J. Biol. Chem., 270: 17060-17063.
• Pawson, T. and Schlessinger, J. (1993). Current Biology, 3: 434-432.
• Picksley, et al. (1994). Oncogene, 9: 2523-2529.
• Pietenpol, et al. (1994). Proc. Natl. Acad. Sci. U.S.A., 91: 1998-2002.
• Pines, J. (1995). Nature, 376: 294-295.
• Polyak, et al. (1994). Cell, 78: 59-66.
• Powis, G. (1992). Trends Pharmacol. Sci., 12: 188-194.
• Rodriguez-Viciana, et al. (1994). Nature, 370: 527-532.
• Sherr, C. (1994). Cell., 79: 551-555.
• Smythe, et al. (1988). EMBO J., 7: 2681-2686.
• Toyoshima, H. and Hunter, T. (1994). Cell, 78: 67-74.
• Waldman, et al. (1995). Cancer Research, 55: 5187-5190.
• Warbrick, et al. (1995). Curr. Biol., 5: 275-282.
• Waga, et al. (1994). Nature, 369: 574-578.
• Yu, et al. (1994). Cell, 76: 933-945.
• Xiong, et al. (1993). Nature, 366: 701-704.
• Zhu, et al. (1995). Genes & Development, 9: 1740-1752.

Claims (41)

Test method for identifying a connection with the ability Interaction or binding between p21 and cyclin D1 and / or cdk 4, wherein the method comprises: (A) contacting: (i) a substance containing a peptide fragment of p21, which consists of an amino acid sequence that is selected out: RERWNFDFVTETPLEGDFAW (peptide 4) KACRRLFGPVDSEQLSRDCD (Peptide 2) KxxRRyFzP (where x can be any amino acid, y and z can be hydrophobic and each of the underlined residues is missing or different can) KRRQTSMTDFYHSKRRLIFS (peptide 10) KRRQTSATDFYHSKRRLIFS KRRQTSMTAFYHSKRRLIFS TSMTDFYHSKRRLIFSKRKP (Peptide 11) KRRLIFSK or xyLzF (where y and z are any amino acids are and x is preferably R), or such a fragment, linked to a binding partner or fused to a non-p21 sequence; and (ii) a substance comprising cyclin D1 and / or cdk4, or a derivative or analog thereof, wherein the derivative cyclin D1 and / or cdk4 activity, and a test compound under conditions, these substances being in Absence of the test compound which is an inhibitor of interaction or Bonding of the substances is, interacts or binds to each other; and (b) determining interaction or binding between the substances. The test method of claim 1, wherein the fragment of p21 from the amino acid sequence consists of peptide 4. The test method of claim 1, wherein the fragment of p21 from the amino acid sequence KxxRRyFzP exists. The test method of claim 1, wherein the fragment of p21 from the amino acid sequence consists of peptide 2. The test method of claim 1, wherein the fragment of p21 from the amino acid sequence xyLzF exists. The test method of claim 1, wherein the fragment of p21 from the amino acid sequence consists of peptide 10. The test method of claim 1, wherein the fragment of p21 from the amino acid sequence KRRLIFSK consists. The test method of claim 7, wherein the fragment of p21 from the amino acid sequence consists of peptide 11. Test method for identifying a connection with the ability Interaction or binding between p21 and cyclin. D1 and / or cdk 4, wherein the method comprises: (A) contacting: (i) a substance containing a peptide fragment from 40 or fewer amino acids of p21, or a derivative of such a fragment, wherein the Fragment or derivative an amino acid sequence: KxxRRyFzP (where x is any amino acid y and z can be hydrophobic and each of the underlined Remains are missing or may be different) includes, where the derivative has the property of attaching to cyclin D1 and / or cdk4 or to interact with it, or such a fragment or De rivat, bound to a binding partner or fused to a Non-p21 sequence; and (ii) a substance containing cyclin D1 and / or cdk4, or a derivative or analog thereof, wherein the Derivative retains cyclin D1 and / or cdk4 activity, and a test compound under conditions which, in the absence of the test compound, which is an inhibitor of interaction or binding of the substances is, interacts or binds to each other; and (b) the Determining interaction or binding between substances. A method according to any one of claims 1 to 9, wherein a compound additionally tested for the ability will modulate a p21-mediated effect on cdk4 activity. The method of claim 10, wherein the cdk4 activity is Rb phosphorylation includes. The method of claim 10, wherein the induction is tested by G1 cell cycle arrest. Use in screening for compounds capable of modulating interaction or binding between p21 and cyclin D1 and / or cdk4, a substance which is a peptide fragment of p21 consisting of an amino acid sequence selected from: RERWNFDFVTETPLEGDFAW ( Peptide 4) KACRRLFGPVDSEQLSRDCD (peptide 2) KxxRRyFzP (where x may be any amino acid, y and z may be hydrophobic, and any of the underlined residues may be missing or different) KRRQTSMTDFYHSKRRLIFS (peptide 10) KRRQTSATDFYHSKRRLIFS KRRQTSMTAFYHSKRRLIFS TSMTDFYHSKRRLIFSKRKP (peptide 11) KRRLIFSK or xyLzF (wherein y and z are any amino acids and x is preferably R), or such a fragment bound to a binding partner or fused to a non-p21 sequence. Use in screening for compounds found in are capable of interaction or binding between p21 and cyclin D1 and / or cdk4, a substance that makes up a peptide fragment 40 or less amino acids of p21, or a derivative of such a fragment, wherein the fragment or derivative has an amino acid sequence KxxRRyFzP (where x any amino acid y and z can be hydrophobic and each of the underlined Remains are missing or may be different) includes, where the derivative has the property of attaching to cyclin D1 and / or cdk4 or to interact with it, or such a fragment or Derivative bound to a binding partner or fused to a Non-p21 sequence. Use in screening for compounds found in capable of being a p21-mediated Effect on cdk4 activity to modulate a substance that is a peptide fragment of p21, that from an amino acid sequence exists, the selected is from: RERWNFDFVTETPLEGDFAW (peptide 4) KACRRLFGPVDSEQLSRDCD (Peptide 2) KxxRRyFzP (where x can be any amino acid, y and z can be hydrophobic and each of the underlined residues is missing or different can) KRRQTSMTDFYHSKRRLIFS (peptide 10) KRRQTSATDFYHSKRRLIFS TSMTDFYHSKRRLIFSKRKP (Peptide 11) KRRLIFSK or xyLzF (where y and z are any amino acids are and x is preferably R), or such a fragment, bound to a binding partner or fused to a non-p21 sequence. Use in screening for compounds found in capable of being a p21-mediated Effect on cdk4 activity to modulate a substance containing a peptide fragment of 40 or less amino acids of p21, or a derivative of such a fragment, wherein the fragment or derivative has an amino acid sequence KxxRRyFzP (where x any amino acid y and z can be hydrophobic and each of the underlined Remains are missing or may be different) includes, where the derivative has the property of attaching to cyclin D1 and / or cdk4 or to interact with it, or such a fragment or Derivative bound to a binding partner or fused to a Non-p21 sequence. Use according to claim 15 or claim 16, wherein the cdk4 activity Rb phosphorylation includes. Use according to claim 13, wherein the induction is tested by G1 cell cycle arrest. Use according to claim 13 or claim 15 wherein the p21 fragment from the amino acid sequence consists of peptide 4. Use according to claim 13 or claim 15 wherein the p21 fragment from the amino acid sequence KxxRRyFzP exists. Use according to claim 13 or claim 15 wherein the p21 fragment from the amino acid sequence consists of peptide 2. Use according to claim 13 or claim 15 wherein the p21 fragment from the amino acid sequence xyLzF exists. Use according to claim 13 or claim 15 wherein the p21 fragment from the amino acid sequence consists of peptide 10. Use according to claim 13 or claim 15 wherein the p21 fragment from the amino acid sequence KRRLIFSK exists. Use according to claim 13 or claim 15 wherein the p21 fragment. from amino acid sequence consists of peptide 11. Use of a substance which is a peptide having the sequence KRRQTSATDFYHSKRRLIFS KRRQTSMTAFYHSKRRLIFS TSMTDFYHSKRRLIFSKRKP (peptide 11) or KRRLIFSK or a fragment thereof of at least 5 amino acids; or a peptide fragment of p21 consisting of an amino acid sequence selected from: RERWNFDFVTETPLEGDFAW (peptide 4) KACRRLFGPVDSEQLSRDCD (peptide 2) KxxRRyFzP (where x can be any amino acid, y and z can be hydrophobic and any of the underlined residues are absent or may be different), or such a fragment or derivative bound to a binding partner or fused to a non-p21 sequence, for the preparation of a drug for the treatment of hyperproliferative disease. Use according to claim 26, wherein the fragment or derivative, a peptide fragment having a sequence which KRRQTSATDFYHSKRRLIFS KRRQTSMTAFYHSKRRLIFS TSMTDFYHSKRRLIFSKRKP (Peptide 11) or KRRLIFSK is, includes or consists of. Use according to claim 27, wherein the substance consists of the peptide KRRLIFSK. Use according to any one of claims 26 to 28, wherein the substance to a carrier for the Feed is bound to cells. Use according to claim 29, wherein the substance is a peptide and attached to a carrier peptide is bound to the sequence RQIKIWFQNRRMKWKK. Peptide KRRLIFSK with the property to inhibit cdk4, for use in a method of treating the human body or carcass by means of therapy. The peptide of claim 31, wherein the treatment is that a hyperproliferative disorder. In vitro or ex vivo method of disturbing the Interaction between p21 and cyclin D1 and / or cdk4, wherein the Method of contacting p21 and / or cdk4 with a substance which is a peptide fragment of p21 derived from an amino acid sequence exists, the selected is from: RERWNFDFVTETPLEGDFAW (peptide 4) KACRRLFGPVDSEQLSRDCD (Peptide 2) KxxRRyFzP (where x can be any amino acid, y and z can be hydrophobic and each of the underlined residues is missing or different can) KRRQTSMTDFYHSKRRLIFS (peptide 10) KRRQTSATDFYHSKRRLIFS KRRQTSMTAFYHSKRRLIFS TSMTDFYHSKRRLIFSKRKP (Peptide 11) KRRLIFSK or xyLzF (where y and z are any amino acids are and x is preferably R), or such a fragment, bound to a binding partner or fused to a non-p21 sequence, includes. In vitro or ex vivo method of modulation a p21-mediated effect on cdk4 activity, the method Contacting p21 and / or cdk4 with a substance containing a peptide fragment of p21, which consists of an amino acid sequence that is selected out: RERWNFDFVTETPLEGDFAW (peptide 4) KACRRLFGPVDSEQLSRDCD (Peptide 2) KxxRRyFzP (where x can be any amino acid, y and z can be hydrophobic and each of the underlined residues is missing or different can) KRRQTSMTDFYHSKRRLIFS (peptide 10) KRRQTSATDFYHSKRRLIFS KRRQTSMTAFYHSKRRLIFS TSMTDFYHSKRRLIFSKRKP (Peptide 11) KRRLIFSK or xyLzF (where y and z are any amino acids are and x is preferably R), or such a fragment, bound to a binding partner or fused to a non-p21 sequence, includes. Use of nucleic acid for a substance that is a peptide mild sequence KRRQTSATDFYHSKRRLIFS KRRQTSMTAFYHSKRRLIFS TSMTDFYHSKRRLIFSKRKP (Peptide 11) or KRRLIFSK or a fragment thereof at least 5 amino acids is; or a peptide fragment of p21 derived from an amino acid sequence exists that is selected out: RERWNFDFVTETPLEGDFAW (peptide 4) KACRRLFGPVDSEQLSRDCD (Peptide 2) KxxRRyFzP (where x can be any amino acid, y and z can be hydrophobic and each of the underlined residues is missing or different can) or such a fragment bound to a binding partner or fused to a non-p21 sequence encoded for production a medicament for the treatment of hyperproliferative disease Expression in a target cell. Use according to claim 32, wherein the fragment or derivative, a peptide fragment having a sequence which RERWNFDFVTETPLEGDFAW (Peptide 4) KACRRLFGPVDSEQLSRDCD (peptide 2) KRRQTSATDFYHSKRRLIFS KRRQTSMTAFYHSKRRLIFS TSMTDFYHSKRRLIFSKRKP (Peptide 11) or KRRLIFSK is, includes or consists of. Use of a substance which is a peptide fragment of p21 consisting of an amino acid sequence selected from: RERWNFDFVTETPLEGDFAW (peptide 4) KACRRLFGPVDSEQLSRDCD (peptide 2) KxxRRyFzP (where x can be any amino acid, y and z can be hydrophobic and each the underlined residues may be missing or different) KRRQTSMTDFYHSKRRLIFS (peptide 10) KRRQTSATDFYHSKRRLIFS KRRQTSMTAFYHSKRRLIFS TSMTDFYHSKRRLIFSKRKP (peptide 11) KRRLIFSK or xyLzF (wherein y and z are any amino acids and x is preferably R), or such a fragment bound to a binding partner or fused to a non-p21 sequence when designing a functional mimetic of p21 having the property of binding to or interacting with cyclin D1 and / or cdk4 and / or screening thereon. In vitro or ex vivo method of disturbing the Interaction between p21 and cyclin D1 and / or cdk4, wherein the Method of contacting p21 and / or cdk4 with a substance which is a peptide fragment of 40 or fewer amino acids of p21, or a derivative of such a fragment, wherein the fragment or derivative an amino acid sequence KxxRRyFzP (where x is any amino acid y and z can be hydrophobic and each of the underlined Remains are missing or may be different) includes, where the derivative has the property of attaching to cyclin D1 and / or cdk4 or to interact with it, or such a fragment or Derivative bound to a binding partner or fused to a Non-p21 sequence, includes. In vitro or ex vivo method of modulation a p21-mediated effect on cdk4 activity, the method Contacting p21 and / or cdk4 with a substance containing a peptide fragment from 40 or fewer amino acids of p21, or a derivative of such a fragment, wherein the Fragment or derivative an amino acid sequence KxxRRyFzP (where x any amino acid y and z can be hydrophobic and each of the underlined Remains are missing or may be different) includes, where the derivative has the property of attaching to cyclin D1 and / or cdk4 or to interact with it, or such a fragment or Derivative bound to a binding partner or fused to a Non-p21 sequence, includes. Use of nucleic acid necessary for a substance containing a peptide fragment from 40 or fewer amino acids of p21, or a derivative of such a fragment, wherein the Fragment or derivative an amino acid sequence KxxRRyFzP (where x any amino acid y and z can be hydrophobic and each of the underlined Remains are missing or may be different) includes, where the derivative has the property of attaching to cyclin D1 and / or cdk4 to bind or interact with, or such a fragment or derivative bound to a binding partner or fused to a non-p21 sequence encoded for the manufacture of a medicament for the treatment of hyperproliferative disease by expression in a target cell. Use of a substance containing a peptide fragment from 40 or fewer amino acids of p21, or a derivative of such a fragment, wherein the fragment or derivative has an amino acid sequence KxxRRyFzP (where x any amino acid y and z can be hydrophobic and each of the underlined Remains are missing or may be different) which comprises the derivative has the property of attaching to cyclin D1 and / or cdk4 or to interact with it, or such a fragment or Derivative bound to a binding partner or fused to a Non-p21 sequence, when designing a functional mimetic of p21 with the property of binding to cyclin D1 and / or cdk4 or to interact with it, and / or when screening on it.